This study aimed to determine the allele frequencies and genetic diversity of 21 autosomal short tandem repeat (STR) loci from the Expanded U.S. Core Loci and European Standard Set in the Romanian population. A random sample of 928 unrelated men from all Romanian counties was analyzed using the Investigator 24plex QS and Investigator 24plex GO! Kits (Qiagen). The genotypes were determined, and the allele frequencies were calculated using the STRidER tool. The results provide updated population genetic data for the Romanian population, which is essential for accurate calculation of DNA evidence weight in forensic casework.

Y-chromosomal Short Tandem Repeats (Y-STRs) provide insight into forensic evidence interpretation and male population history. Romania’s position at the crossroads of Europe and Western Asia makes it a key reference population for both domains, yet existing Romanian Y-STR databases are limited in geographic coverage and marker number. We genotyped 928 unrelated Romanian males across all 41 counties and Bucharest at 27 Y-STR loci using the Investigator Argus Y-28 Kit, and modeled haplotype frequencies using the Discrete Laplace (DL) distribution (disclapmix v1.7.5, 17-locus panel; disclapmix2 v0.6.1, 27-locus panel). Discrete Laplace modeling revealed 12 and 14 optimal mixture components for the 17- and 27-locus models, respectively, with median match probabilities of 1 in 1.25 × 10⁷ and 1 in 5.8 × 10¹², reflecting nearly six order-of-magnitude gain in evidential weight. Haplogroup analysis identified 16 Y-lineages; R1a, I2a, E1b1b, J2a, and R1b collectively represent ~80% of the population, and reflect Romania’s layered Steppe, Balkan, and Near Eastern ancestry, with subtle regional gradients between Moldavia, Transylvania, and Muntenia. This dataset provides validated forensic reference data and new insights into the patrilineal genetic structure of the Romanian population.