Hi..
 
A probable 99% test for queenlesness would be to put a frame of brood containing eggs or
24hrs old larvae into the suspect hive..(be carefull that you dont transfer disease here).   If
queenless then they will produce emergency Q cells on the face of the comb.
Beware, sometimes these cells will produce small runty Qs that produce a small brood nest.
 
Regards
Phil