Acid lime [Citrus aurantifolia (Christm.) Swingle] is a fruit crop, enriched with high commercial value and is cultivated in 60 out of 75 districts representing all geographical landscapes of Nepal. Lack of high yielding cultivars is probably one of the main reason for its extremely reduced productivity which warrants a deep understanding of genetic diversity in existing germplasm. Hereby, we aim to access the genetic diversity of acid lime germplasm cultivated at 3-different ecological gradients of eastern Nepal employing PCR-based Inter-Simple Sequence Repeats markers (ISSR). Altogether, 21 polymorphic ISSR markers were used to assess the genetic diversity in 60 acid lime cultivars sampled from different geographical locations. Analysis of binary data matrix was performed on the basis of bands obtained, scoring of the data was done accordingly, and principal coordinate analysis and phenogram were constructed using different computer algorithms. ISSR profiling yielded 234 amplicons, of which 87.18% were found to be polymorphic. The number of amplified fragments ranged from 7-18 with amplicon size ranging from 250-3200 bp. The NTSYS based Cluster analysis using UPGMA algorithm taking Dice Similarity coefficient separated 60 accessions into 2-major and 3-minor clusters. The genetic diversity analysis revealed the highest for Terai and the lowest for High-hill zone. Cluster I comprised of accessions from High-hill and Mid-hill regions revealing the close genetic relationship, whereas cluster II comprised of accessions from all three agro-ecological zones and the exotic varieties. Furthermore, our results revealed the accessions harvested from different geographical gradients were not genetically distinct, but highest diversity was observed in Terai accessions in comparison to the regions belonging to the High and Mid-hills. Thus, our data indicate that the ISSR provides a better option for evaluating the genetic diversity of Nepalese Acid Lime cultivars and furnished significant information, assisting parental selection in current and future breeding programs and germplasm conservation which ultimately may help to provide a technological breakthrough for the farmers of the developing country like Nepal.