Understanding the genetic structure of domestic species provides a window into the process of domestication. This study attempts to offer an insight into the prevailing genetic status of Tunisian indigenous rabbit breeds using molecular markers. Thirty-six microsatellite loci were used to provide a comprehensive insight into the genetic status and relationship among 12 Tunisian indigenous rabbit populations. A total of 264 rabbits from villages of the Tozeur and Kebili regions were studied. Standard statistics parameters of genetic variability within and between populations were calculated. The observed heterozygosity, unbiased expected heterozygosity and the effective number of alleles were used to assess the genetic variation of each indigenous breed. Results show a high genetic diversity and observed heterozygosity ranged between 0.3 and 0.5, which implies that there is an abundant genetic variation stored in Tunisian indigenous rabbit breeds. Significant population differentiation was observed (F-ST=0.11), which means that most of the genetic variation resides within breeds. The percentage of individuals correctly classified to their population was 85%. Breeds with more than one breeder origin were divided into subgroups, due to differences in gene frequencies between breeders, which in some cases creates a genetic differentiation even higher than that observed between distinct breeds. The current study is the first detailed analysis of the genetic diversity of Tunisian indigenous rabbit populations. The data generated here provides valuable information about the genetic structure of the 12 rabbit populations and this can be used to designate priorities for their conservation.