Twenty ISSR markers were screened to characterize genetic diversity present among 23 F3 interspecific progenies and their parents. Out of twenty primers, used fifteen primers showed amplification. A total of 79 ISSR loci were detected, out of which, 66 loci were polymorphic. The numbers of amplified loci varied from three (Primer 4824-040, 4824-047 and 4824-48) to a maximum of eight (Primer 4824-043, 4824-044 and 4824-055), with a size range of 250-2100 bp. The average number of bands per primer and polymorphic bands per primer were 5.26 and 4.4, respectively. Percentage of polymorphism ranged from 33% (Primer 4824-048) to 100% (Primer 4824-038, 4824-041, 4824-049 and 4824-055) with mean polymorphic information content (PIC) value of 0.85. Progenies with high molecular diversity could be used in breeding programmes and development of heterotic gene pools with broad genetic base. ISSR marker system was found to be useful for the genetic diversity studies in pigeonpea and to study variation within segregating population.