Genetic analysis of Y-STR loci is pivotal for forensic libraries and genetic analysis. The Koli population in Gujarat, India, however, lacks such genetic characterization. This study aims to develop an allele frequency database for 23 Y-STR loci in the Koli population, examining forensic parameters and assessing genetic connections with neighboring tribes. A total of 153 unrelated Koli males were genotyped using the PowerPlex®Y23 multiplex commercial kit. We identified 117 distinct haplotypes. The Haplotype Diversity (HD) and Discrimination Capacity (DC) for the 23 Y-STR loci were 0.993 and 0.8034, respectively. DYS385b locus exhibited the highest allele variability (10 alleles), whereas DYS391, DYS389I, and DYS437 showed the least (4 alleles each). The highest Polymorphic Information Content (PIC) was observed in DYS385b (0.775), with the lowest in DYS391 (0.386). The dominant haplogroup R1a accounted for 45% of the population. Comparative analysis with other Indian populations from YHRD revealed two