Introduction: Annona squamosa L. commonly known as custard apple, is an important under-utilized fruit species from tropical and subtropical parts of India with high potential as commercial horticultural crop. The aim of this study was to determine the genetic diversity among populations spread across districts of Udaipur, Rajsamand and Chittaurgarh falling within Aravallis in Rajasthan. In all, 21 populations were identified and subjected to molecular evaluation using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers. The cumulative analysis carried out on the basis of RAPD and ISSR data sets revealed 73.91% polymorphism. Methods: A total of 251 amplicons were produced using 19 RAPD and 15 ISSR primers. The pair-wise distance matrix calculated by Dice’s co-efficient showed a distance range of 0.67 to 0.95 when computed using cumulative data set. Results: Characterization of genetic diversity within populations based on combined data set showed that percentage of polymorphism bands (PPB) ranged from 29.19 to 50.93. Nei’s gene diversity (HE) values varied from 0.1002 to 0.1964 within a total genetic diversity (HT) of 0.2509. The average observed number of alleles per locus (Na) was 1.3602 and Ne was 1.2400. The Shanon’s index was 0.2002 (Hpop) and 0.3800 (Hsp) at population and species level, respectively. Conclusion: The moderate gene flow value (Nm=0.59) and significant coefficient of genetic differentiation (GST=0.458) revealed low level of genetic diversity within population and highly significant genetic differentiation among populations of A. squamosa. The UPGMA dendrogram resolved all the 21 populations representing 3 districts into various clusters according to their genetic distances. The principal component analysis (PCA) supported the UPGMA results and justified the relationship among populations.