Inbreeding may have important consequences for the genetic structure of social insects and thus for sex ratios and the evolution of sociality and multiple queen (polygynous) colonies. The influence of kinship on mating preferences was investigated in a polygynous ant species, Iridomyrmex humilis, which has within-nest mating. When females were presented simultaneously with a brother that had been reared in the same colony until the pupal stage and an unrelated male produced in another colony, females mated preferentially with the unrelated male. The role of environmental colony-derived cues was tested in a second experiment where females were presented with two unrelated males, one of which had been reared in the same colony until the pupal stage (i.e., as in the previous experiment), while the other had been produced in another colony. In this experiment there was no preferential mating with familiar or unfamiliar males, suggesting that colony-derived cues might not be important in mating preferences. Inbreeding was shown to have no strong effect on the reproductive output of queens as measured by the number of worker and sexual pupae produced. The level of fluctuating asymmetry of workers produced by inbreeding queens was not significantly higher than that of non-inbreeding queens. Finally, colonies headed by inbreeding queens did not produce adult diploid males. Based on the current hypotheses of sex-determination the most plausible explanations for the absence of diploid-male-producing colonies are that (i) workers recognized and eliminated these males early in their development, and/or (ii) there are multiple sex-determining loci in this species. It is suggested that even if inbreeding effects on colony productivity are absent or low, incest avoidance mechanisms may have evolved and been maintained if inbreeding queens produce a higher proportion of unaviable off-spring.