I can do it reliably in 7 Questions (improved 6 Question answer below):
Q1-4 Will the next person tell me the truth?
There must be at least one Pass, possibly 2
In the following, R = Riddler, P = Penguin, T = TwoFace, J = Joker
R,P,T must Pass if J is next
R,P must Pass if T or J is next
if J is next after T there will be 2 Passes together
if T is after J, there will only be 1 Pass
Neither P nor R will answer Yes
We now have some idea of the order:
if there are two Passes one after the other, then T immediately precedes J
if there is only one Pass, then J immediately precedes T
in both cases above, R and P are together
if two Passes are not together, then R and P are not together (they both Passed),
so R and P are alternating with T and J
It is really only the Passes (Z) which are significant above, After 4 questions, we have these possibilities:
In the following, in addition to the notation above, Z = Pass, x = Yes or No, ? = R or P, # = T or J
1. TwoFace followed by Joker
ZZxx == ?TJ? .Q5 Are you the Joker? (Y = PTJR )
xZZx == ??TJ .Q5 Are you the Joker? (Y = PRTJ )
xxZZ == J??T .Q5 Are you the Joker? .Q6 Are you the Joker?
ZxxZ == TJ?? .Q5 Are you the Joker? .Q6 Are you the Joker? .Q7 Are you the Joker?
Basically, repeat the question 'Are You The Joker?' until you get an answer from either R or P, The Riddler will answer 'No', the Penguin will answer 'Yes'
2. Joker followed by TwoFace
Pxxx == ?JT? .Q5 Are you the Joker?
xPxx == ??JT .Q5 Are you the Joker?
xxPx == T??J .Q5 Are you the Joker? .Q6 Are you the Joker?
xxxP == JT?? .Q5 Are you the Joker? .Q6 Are you the Joker? .Q7 Are you the Joker?
The same process as above.
3. Riddler and Penguin alternating with Joker and TwoFace
( ? = R or P, # = T or J )
PxPx == ?#?# .Q5 Are you the Joker? .Q6 Will the Joker tell me the truth?
xPxP == #?#? .Q5 Will the Joker tell me the truth? .Q6 Are you the Joker?
In this case, it is slightly trickier than the above two cases, because we only know TJ and RP are alternating, but we do not know how. So we need to ask the following two questions in the right order:
For Penguin or Riddler: Are you the Joker? Riddler will answer 'No', Penguin will answer 'Yes'
For TwoFace or Joker: Will the Joker tell me the truth? Two-Face will Pass, Joker will answer Yes or No
Maximum 7 Questions (but I think it should be possible to do it in less)
Bonus, No Villain gets asked the same question twice.
EDIT Improved answer below - 6 questions:
Ask the following question four times:
If I ask the next person if they are the Riddler, will they say 'Yes'?
If the Riddler answers the question and the next person is the Penquin, he will say 'Yes' otherwise he will 'Pass'.
If the Penquin answers the question and the next person is the Riddler, he will say 'No', otherwise he will 'Pass'
If TwoFace answers the question, he will 'Pass' if the next person is the Joker, otherwise he will answer 'Yes' or 'No'
If Joker answers the question, he will just answer 'Yes' or 'No'
This will tell us some thing about the order and might actually tell us what the order is:
If the Joker follows TwoFace, then we will have two Passes together, the first from either the Riddler or the Penguin, and the second from TwoFace.
The previous answer, before the first 'Pass' will tell us whether the Penguin is before or after the Riddler, e.g.
Yes,Pass,Pass,No - would be Riddler, Penguin, TwoFace, Joker
and ...
No,Pass,Pass,No - would be Penguin, Riddler, TwoFace, Joker
alternatively:
If TwoFace follows the Joker, then there will be only 1 'Pass' (the Penguin or the Riddler) which will identify the Joker, but TwoFace will be the next person, so:
Yes,Pass,No,Yes - would be Riddler, Penguin, Joker, TwoFace
Likewise,
No,Yes,Pass,No - would be TwoFace, Riddler, Penguin, Joker
So anything ordered similarly to the above will only need 4 questions.
Otherwise, the Riddler and the Penguin will be alternating with TwoFace and Joker, and there will be two Passes, one from the Riddler, and one from the Penguin. So in this case it is simply a matter of telling the Joker from TwoFace, after the second 'Pass' answer is given - and then telling the Riddler from the Penguin
If you ask TwoFace/Joker, 'Will the Joker tell me the truth?', TwoFace must always 'Pass', the Joker must answer 'Yes' or 'No'
If you ask the Riddler/Penguin, 'Will the Penguin tell me the truth?', the Riddler will always answer 'No', the Penguin will always answer 'Yes'.
Bonus: No one will be asked the same question twice.
So, Batman can work the order out in a maximum of 6 questions.