Ustilago segetum (Pers.) Roussel tritici (UST) causes loose smut of wheat account for considerable grain yield losses globally. For effective management, knowledge of its genetic variability and population structure is a prerequisite. In this study, UST isolates sampled from four different wheat growing zones of India were analyzed using the second largest subunit of the RNA polymerase II (RPB2) and a set of sixteen neutral simple sequence repeats (SSRs) markers. Among the 112 UST isolates genotyped, 98 haplotypes were identified. All the isolates were categorized into two groups (K = 2), each consisting of isolates from different sampling sites, on the basis of unweighted paired-grouping method with arithmetic averages (UPGMA) and the Bayesian analysis of population structure. The positive and significant index of association (I_A = 1.169) and standardized index of association (rBar_D = 0.075) indicate population is of non-random mating type. Analysis of molecular variance showed that the highest variance component is among isolates (91%), with significantly low genetic differentiation variation among regions (8%) (F_st = 0.012). Recombination (R_m = 0) was not detected. The results showed that UST isolates have a clonal genetic structure with limited genetic differentiation and human arbitrated gene flow and mutations are the prime evolutionary processes determining its genetic structure. These findings will be helpful in devising management strategy especially for selection and breeding of resistant wheat cultivars.