Reproductive conflicts that arise in multiple-queen (polygynous) social insect colonies often lead to behavioural dominance by one queen. No evidence of dominance was found in an intensive study of queen behaviour patterns in colonies of the facultatively polygynous ant Myrmica tahoensis. Colonies were monitored in artificial nests for 228 hours (432 queen-hours). Observations were divided between seven monogynous colonies, nine polygynous colonies, and seven colonies containing one native queen and an unrelated queen that had recently been transplanted into the colony. Aggression rarely occurred between queens in the polygynous colonies, virtually all queens laid eggs, and there was no sign of skew in egg-laying rates. Overall queen activity levels varied considerably between colonies in the three colony types. Queens in monogynous colonies spent more time antennating workers and were groomed more than polygynous queens, whereas polygynous queens laid eggs, handled brood and moved more often. Introduced queens were groomed at a higher rate than queens in naturally polygynous colonies, and queens from naturally polygynous colonies antennated workers, moved, laid eggs and antennated other queens more frequently. Within the introduced-queen colonies, native queens laid eggs and handled brood more frequently than introduced queens. Following the behavioural study, queen–queen relatedness was estimated within the polygynous colonies by means of microsatellite DNA polymorphisms. Queen behaviour patterns were similar regardless of whether queens were close or distant relatives. These results suggest that queens and workers of M. tahoensis behave differently towards newly introduced queens, but do not respond to queen–queen relatedness levels.