Azadirachtin, a key bioactive compound derived from the neem tree (Azadirachta indica A. Juss), holds paramount importance in various industrial applications due to its potent pesticidal properties and versatile nature. The existence of natural variability for azadirachtin among different Neem genotypes presents an opportunity for strategic selection and breeding. The variability observed across genotypes allows for the identification and breeding of genotypes consistently high in azadirachtin content. Genetic diversity studies of A. indica using Inter Simple Sequence Repeats (ISSR) markers revealed that the 40 genotypes were grouped into 7 distinct clusters based on Jaccards’ similarity coefficient. The dendrogram obtained at a 0.73 similarity matrix showed the highest genetic diversity between genotypes G1 and G25, in contrary to this, G32 and G37 showed the least genetic diversity. A total of 33 primers were used in the study, but only 20 primers were successfully amplified at different annealing temperatures, with the Polymorphism Information Content (PIC) values ranging from 0.201 to 0.385, averaging 0.31. Most of the primers exhibited 100 % polymorphism. The number of bands generated ranged from 6–14. Among all the bands generated, 13 unique bands were identified across 12 primers in 14 genotypes, with G16 and G25 showing the highest number of unique bands. These findings provide valuable insights for selecting superior genotypes as candidate trees for future improvement programs, contributing to better management and commercial utilisation of neem resources. This study also plays a crucial role in advancing the genetic enhancement of A. indica.