In the present investigation 94 recombinant inbred lines from wheat cross Aldan (resistant) and WH542 (susceptible), were used. There was significant variation among all the recombinant inbred lines for coefficient of infection of Karnal bunt (Neovossia indica). The chi-square analysis indicated that a single dominant gene was responsible for Karnal bunt resistance. Total thirty five inter simple sequence repeat (ISSR) primers were used for amplification of DNA from the two parents. ISSR markers based finger print database were generated using only 25 primers. Out of 25, six primers showed 100% polymorphism. A total of 193 alleles were detected at 25 ISSR loci. The number of alleles per locus ranged from 4-14 with an average of 7.72 alleles per locus. The two parents were genetically diverse (similarity coefficient 0.419). The cluster analysis led to distribution into two groups: group 1 had resistant parent Aldan and group 2 had susceptible parent WH542, resistant bulk and susceptible bulk respectively. Among all the primers used one primer (IS-43) was identified to be associated with Karnal bunt resistance. This showed an amplification profile (650 bp product) characteristic of resistant parent, in corresponding bulk and in individual genotyping of the recombinant inbred lines. This might have an application in marker assisted selection after converting it to SCAR marker.