A robust Genotyping-By-Sequencing (GBS) pipeline platform was examined to provide accurate discovery of Single Nucleotide Polymorphisms (SNPs) in a cape gooseberry (Physalis peruviana L.) and related taxa germplasm collection. A total of 176 accessions representing, wild, weedy, and commercial cultivars as well as related taxa from the Colombian germplasm bank and other world repositories were screened using GBS. The pipeline parameters mnLCov of 0.5 and a mnScov of 0.7, tomato and potato genomes, and cape gooseberry transcriptome for read alignments, were selected to better assess diversity and population structure in cape gooseberry and related taxa. A total of 7,425 SNPs, derived from P. peruviana common tags (unique 64 bp sequences shared between selected species), were used. Within P. peruviana, five subpopulations with a high genetic diversity and allele fixation (HE: 0.35 to 0.36 and FIS: -0.11 to -0.01, respectively) were detected. Conversely, low genetic differentiation (FST: 0.01 to 0.05) was also observed, indicating a high gene flow among subpopulations. These results contribute to the establishment of adequate conservation and breeding strategies for Cape gooseberry and closely related Physalis species.