Natural selection should optimize litter size in response to the distribution and abundance of resources during breeding. In semelparous, litter-bearing antechinuses, teat number limits litter size. Consequently adaptation has been inferred in explaining intraspecific, geographic variability in teat number for several Antechinus spp. The phylogeography of teat number variation and associated genetic divergence were assessed in A. agilis using nine microsatellites and mitochondrial cytochrome b sequence data. Six-teat Otway Range animals were divergent in microsatellite allele identity and frequencies: samples from three Otway six-teat sites demonstrated significantly greater similarity genetically to those from six-teat animals ∼250 km to the west, than to nearby Otway 10-teat samples, or to the six-teat animals at Wilsons Promontory. Gene flow between Otway phenotypes appears to have been limited for sufficient time to enable different microsatellite alleles to evolve. Nonetheless, nuclear genetic evidence suggested only incomplete reproductive isolation, and mitochondrial DNA (mtDNA) haplotypes showed no association with teat number. Other populations across the range were no more genetically differentiated from one another than expected from geographic separation. Principal components and distance-based redundancy analyses found an association between environmental variables and geographic distribution of A. agilis teat number — six-teat animals inhabit more temperate forests, whilst those with more teats experience greater seasonality. The apparent restricted breeding between phenotypically distinct animals, together with phylogenetically separate groups of six-teat animals in different locations with similar environments, are consistent with the hypothesis that adaptation to different habitats drives teat number variation in A. agilis.