The genus Pogonomyrmex is one of three ant genera with an effective mating frequency (me) > 2.0. We developed microsatellites to determine me for P. rugosus because mating frequency of P. rugosus was known only from observational data which do not allow an estimate of me. We genotyped 474 workers from 20 colonies for two microsatellite loci. Observed mating frequencies ranged from 3 to 12 and me for P. rugosus was 4.71. Observed patriline frequencies were significantly different from the expected patriline frequencies generated with a simulated data set under the assumption of equal patriline representation. The available mating frequency data and phylogenetic information of the genus Pogonomyrmex suggest that multiple mating is the ancestral state in the North American Pogonomyrmex sensu stricto. Established P. rugosus colonies raid and destroy smaller conspecific colonies. During these raids ant workers were observed carrying pupae and larvae from the raided colony into the nest of the raiding colony. However, it was not clear whether raided brood emerged in the raiding colony and were subsequently recruited into the work force (intraspecific slavery) or were used as food (predation). Our analyses indicate 6 of 14 field colonies contained foreign P. rugosus workers (43%). The range of the intracolonial frequency of foreign workers collected directly from the nest entrance was between 4 and 28%.