Tilletia indica is a floret infecting quarantined fungal pathogen of wheat causing Karnal bunt of wheat. The population of Tilletia indica is highly genetically diversed and it is difficult to breed durable and resistant cultivars of wheat. Simple sequence repeats (SSRs) are extensively used to uncover the population structures in fungal pathogens. In present study, a total of 5,772 simple sequence repeat loci were identified in the T. indica genome. In silico analysis, forty microsatellite markers were used to genotype of 20 Tilletia indica isolates from north-western plain zone of India to analyze population structure. SSRs were found in 863 scaffolds. Among SSRs, tri-nucleotide was most abundant (42 %), followed by di-nucleotide (28 %), mono-nucleotide (23 %), tetra-nucleotide (3 %), hexa-nucleotide (3 %) and penta-nucleotide (1 %). 130 alleles were amplified in 40 SSR markers among the 20 T. indica. The polymorphic information value content (PIC) values ranged from 0.20 to 0.81 with an average of 0.51. Maximum PIC (0.81) was obtained for TiSSR34 marker. 18 SSRs were highly informative (PIC ≥ 0.5), 15 SSRs were moderately informative (0.5 >PIC <0.25) and remaining 7 SSRs were less informative marker (PIC<0.25). In cluster analysis, T. indica isolates did not cluster to region wise of isolates. Further, the monosporidial (haploid) population of Tilletia indica was also developed. Ten highly polymorphic SSR markers were amplified in 60 monosporidial lines (ms) of T. indica. Differently amplified monosporidial lines were chosen for monosporidial lines compatibility assay. 27 monosporidial crosses of T. indica were produced Karnal bunt disease reaction on susceptible host (WH542). KB7MS1 x KB18MS5 with 36 % coefficient of infection and KB7MS1 x KB16MS1 (coefficient of infection of 24.17 %) was found most virulent. These haploid monosporidial lines may have different mating types. These ms lines were found in distinct clusters. The newly developed SSR markers will be useful for genetic and population studies. This information could be useful for better management of Karnal bunt of wheat in India.