BACKGROUND: Analyzing Y-chromosome short tandem repeats (Y-STRs) is essential in forensic genetics and population studies. The Yfiler™ Plus kit, which includes 27 Y-STR markers, enhances the discrimination power for forensic and kinship applications. However, this genetic system has not been analyzed in Mexican populations, which limits its application and representativeness in international databases. OBJECTIVES: We wished to examine the genetic diversity and forensic parameters of the 27 Y-STRs included in the YFiler™ Plus kit in two populations from Western Mexico (Jalisco and Michoacán). METHODS: Male DNA samples were amplified using the Yfiler™ Plus kit, followed by a fragment analysis via capillary electrophoresis (CE). The haplotype frequencies and forensic parameters were calculated. The haplogroups of all samples were predicted, and the distribution and percentages of ancestries were determined. The Rst genetic distances, including reference populations, were calculated and graphically represented in a multidimensional scaling (MDS) plot. RESULTS: A total of 224 haplotypes were identified in all of the samples, of which 98.66% corresponded to unique haplotypes. Bi- and tri-allelic patterns were observed in both populations. The observed discriminatory capacity was 98.4% for Jalisco and 98.9% for Michoacán, while the haplotype diversity values were 0.9998 and 0.9997, respectively. The most frequent haplogroup was R1b, followed by Q, representing the European and Native American ancestries, in both populations. CONCLUSIONS: This study is the first to report the haplotype diversity and forensic parameters of the 27 Y-STRs included in the Yfiler™ Plus kit in Mexican populations. These findings confirm the forensic utility of these markers for human identification, biological relationship testing, and criminal investigations, reinforcing their applicability in forensic casework.