Solanum muricatum Aiton, commonly known as “pepino dulce” or “sweet cucumber”, is a native Andean fruit with growing agronomic and nutraceutical importance. Understanding its genetic diversity and structure is essential for conservation and crop improvement strategies. In this study, we employed genotyping by sequencing (GBS) to evaluate the population structure of 96 accessions of S. muricatum collected from seven Peruvian departments: Ayacucho, Cusco, Apurímac, Ica, Lima, Lambayeque, and Cajamarca. GBS generated over 500 million raw reads, from which 9.5 million tag sequences were identified. After stringent filtering, 767,166 tags were retained, and 269 high-quality single-nucleotide polymorphisms (SNPs) with minor allele frequency > 0.01 were used for downstream analysis. Phylogenetic inference based on a neighbor-joining tree grouped the accessions into several clusters, with some degree of geographic association, though admixture among populations was evident. Principal Coordinate Analysis (PCoA) corroborated this pattern, showing a central core of genetic similarity with a few outliers, particularly from Ayacucho. The genetic data revealed substantial diversity within and between populations, suggesting the existence of both shared ancestry and local adaptation. Despite the low number of SNPs retained after filtering, clustering patterns were consistent with field-collected agro-morphological data, indicating that even a reduced SNP dataset can be informative for population differentiation in S. muricatum. These findings highlight the underexploited genetic diversity of Peruvian “pepino dulce” germplasm and reinforce the relevance of ongoing ex situ conservation and pre-breeding efforts undertaken by national universities in Peru. Our results lay a foundation for more detailed studies involving higher-resolution genomics, association mapping, and the development of molecular markers for agronomic traits in this underutilized crop.