Background: Short tandem repeats (STRs) are highly variable sequences present along the human genome, including the Y-chromosome. Y-STRs are exclusive to males, and the haplotypes they define are informative. Objectives: Twenty-six Y-STR loci were genotyped in 252 males from Northern Portugal to characterise Y-chromosome genetic variation using the Investigator Argus Y28 QS Kit. Methods: The kit mentioned was used to amplify male DNA samples, and capillary electrophoresis was used to analyze the fragments. Forensic parameters and haplotype diversity were computed, and samples’ haplogroups were predicted. A multidimensional scaling (MDS) plot was used to graphically represent the R_ST genetic distances, including reference populations. Results: A total of 250 different haplotypes were observed, including 248 unique ones, yielding a very high haplotype diversity (HD = 0.999) and discriminatory power (DP = 0.992). Haplogroup analysis indicated a predominance of R1b (58.7%), followed by E1b1b, I and J, pointing to a population history shaped by Mediterranean and North African gene flow. Comparative analysis between Portugal and 5 other populations showed greater genetic affinity with Spain and Italy, while revealing marked differentiation from Greece, Morocco, and former Portuguese colonies. Conclusions: The results confirm that the Northern Portuguese Population exhibits high Y-STR variability and robust forensic resolution. The dataset was submitted to the YHRD database, enhancing the representation of the Portuguese population and underscoring the value of the 26 locus panel for applications in forensic science, genealogy, and population genetics.