• Haploblepharus is an understudied genus comprising four recognized scyliorhinid species that are endemic to southern Africa. Species identification within this group has historically been problematic due to a high degree of morphological conservatism among congeners, further complicated by the possibility of interspecific hybridization.
• This study describes the development of two microsatellite panels comprising 10 polymorphic markers for the puffadder shyshark, Haploblepharus edwardsii . The markers were characterized in 35 H. edwardsii specimens and tested for cross‐species utility in Haploblepharus fuscus , Haploblepharus pictus , and the more distantly related scyliorhinid Halaelurus natalensis .
• Genetic diversity statistics were estimated for each species, and the presence of population differentiation was tested for in H. edwardsii and H. pictus . Furthermore, interspecific genetic differentiation was examined to infer the potential use of these markers for species identification as well as for detecting signatures of admixture among Haploblepharus species.
• All microsatellite markers were polymorphic in each species, with polymorphism information contents ranging from 0.43 to 0.62. Population differentiation was only evident for H. pictus , where genetic discontinuity was detected among geographically distant sampling sites.
• Statistically significant differentiation (fixation index between populations F ST = 0.091 to 0.382) was found between all species; however, the level of differentiation between H. fuscus and H. pictus was low in comparison and seemingly at a population level rather than at a species level. Species assignment using Bayesian clustering analysis resulted in approximately 57% of 88 genotyped specimens being unambiguously assigned to a distinct genetic cluster that confirmed accurate taxonomic assignment.
• Overall, the low levels of differentiation together with the presence of distinct and admixed genetic clusters suggests a recent divergence and possible contemporary hybridization within the genus Haploblepharus. As such, conservation strategies should be focused on the generic level until such a time that Haploblepharus species can be readily identified.