Mexican Mestizos (admixed) have been poorly studied for short tandem repeats (STRs) included for new human identification (HID) kits, such as the GlobalFiler PCR Amplification kit. Therefore, this kit was analyzed in 784 unrelated volunteers from the city of Tijuana (n = 381) and Sonora state (n = 403) in the northwest region of Mexico. Allele frequencies, forensic parameters, Hardy-Weinberg equilibrium, and linkage equilibrium were estimated or evaluated for 21 autosomal STRs, respectively. For this HID kit, the combined power of discrimination (PD) was > 0.99999999999999 (RMP range = 1.23 to 3.0 × 10-25), and the combined power of exclusion (PE) were 0.999999993 and 0.999999997 in Tijuana city and Sonora state, respectively. Interpopulation analyses based on STRs of the GlobalFiler kit was performed, including four Mexican Native American, one Mexican Mestizo, and four ethnic American populations (USA), previously studied. The low-but significant-differentiation observed among Mexican Mestizos (FST = 0.0969%; p = 0.02584) justifies the creation of STR databases for HID purposes in this country. In brief, results allow the confident use of the GlobalFiler kit for HID purposes in Mestizo population from the Northwest region Mexico.