The Blue-spotted Salamander (Ambystoma laterale) and the Jefferson Salamander (Ambystoma jeffersonianum) are both part of the Jefferson complex that also includes unisexuals of various ploidy levels and genome composition, referred to as genomotypes. Female unisexuals reproduce mainly through gynogenesis, but ploidy elevations, translocations, recombinations, intergenomic exchanges, and genome replacement also have been documented. The objective of this study was to obtain information on the geographical distribution of genomotypes and the distribution of genetic diversity in southern Québec, Canada. To do so, we examined populations of the Jefferson complex from five sites, including one site from a city and the other sites from protected areas. Genetic analysis using mitochondrial DNA and six microsatellite loci were performed. Results revealed that genomotype frequencies vary greatly, because some populations were mainly composed of A. laterale, whereas unisexuals were clearly predominant in other populations. Diploid, triploid, and putative tetraploid unisexuals were found in some of the populations. One of the populations analyzed seems to be composed only of LJ unisexual salamanders, raising questions about the mode of reproduction used in this population. Finally, the population located in an urban environment presents a low genetic diversity, and we propose hypotheses to explain this finding.