Tomato leaf curl virus (ToLCV) is one of the most destructive viral diseases limiting tomato production worldwide. Identification of resistant genotypes and assessment of their genetic diversity are essential for the development of durable resistant cultivars. The present investigation was undertaken to evaluate twenty-two tomato genotypes and varieties for natural ToLCV incidence and to assess their genetic diversity using Inter Simple Sequence Repeat (ISSR) markers. Disease incidence varied significantly among the genotypes, indicating substantial variability for resistance and susceptibility under natural field conditions. The resistant variety Arka Rakshak remained free from disease infection, whereas susceptible genotypes exhibited high disease incidence. Genomic DNA isolated from all genotypes was amplified using ISSR markers. Twenty ISSR primers generated a large number of reproducible bands with a high level of polymorphism, demonstrating the effectiveness of ISSR markers in detecting genetic variation among tomato genotypes. Cluster analysis based on Jaccard’s similarity coefficient grouped the genotypes into distinct clusters corresponding to their genetic relationships and disease response. Resistant genotypes showed closer genetic association with Arka Rakshak, whereas susceptible genotypes clustered with JT-3 and other highly infected cultivars. The study revealed considerable genetic variability among tomato genotypes and demonstrated the usefulness of ISSR markers for identifying resistant genetic resources and supporting tomato breeding programmes aimed at ToLCV resistance.