Background: The utilisation of molecular markers has increased in molecular research recently. Inter-simple sequence repeat (ISSR) markers allow the analysis of genomes without preliminary sequence information, since random primers are used. The purpose of the present study was to evaluate ISSR markers for assessing the genetic diversity of indigenous Anatolian Water Buffaloes reared in Afyon, Konya and Sivas provinces of Turkey, with a view to conservation of the gene resources. Results: The 11 ISSR primers chosen for the analysis revealed a total of 110 bands, of which 76 (69.09%) were polymorphic. Also, genetic similarity, polymorphic information content (PIC), resolving power (R p) and mean resolving power (R p), heterozygosity (H) and Shannon index (I) were calculated as 0.9479-0.9562, 0.35 ± 0.20, 2.73, 0.27, 0.18 ± 0.07 and 0.28 ± 0.11, respectively. Conclusion: The ISSR markers were found to be promising for assessing the genetic diversity in buffalo populations. Potential genetic parameters such as PIC, R p, R p, H and I were effectively used in this study. © 2011 Society of Chemical Industry.