Among the people living in the five deep Southern Thai provinces, Thai-Malay Muslims (MUS) constitute the majority, while the remaining are Thai Buddhists (BUD). Cultural, linguistic and religious differences between these two populations have been previously reported. However, their biological relationship has never been investigated. In this study, we aimed to reveal the genetic structure and genetic affinity between MUS and BUD by analyzing 15 autosomal short tandem repeats. Both distance and model-based clustering methods showed significant genetic homogeneity between these two populations, suggesting a common biological ancestry. After Islamization in this region during the fourteenth century AD, gradual albeit nonstatistically significant genetic changes occurred within these two populations. Cultural barriers possibly influenced these genetic changes. MUS have closer admixture to Malaysian-Malay Muslims than BUD countrywide. Admixture proportions also support certain degree of genetic dissimilarity between the two studied populations, as shown by the unequal genetic contribution from Malaysian-Malay Muslims. Cultural transformation and recent minor genetic admixture are the likely processes that shaped the genetic structure of both MUS and BUD.