The goal of the current study was to determine the high-resolution frequencies of the HLA-DRB1 alleles among the analyzed Romanian cohort of healthy stem cell donors. Using Next Generation Sequencing (NGS), we estimated class II HLA-DRB1 allele frequencies to a 6-digit resolution through HLA typing in a Romanian cohort of healthy individuals. The study for HLA genotyping included 420 willing donors from the National Registry of Voluntary Hematopoietic Stem Cell Donors (RNDVCSH). In 2020 and 2021, peripheral blood samples were collected and transported to the Fundeni Clinical Institute. We used the Immucor Mia Fora NGS MFlex kit for HLA genotyping. Forty-one different alleles were detected in 420 analyzed samples, out of which the most frequent HLA-DRB1 alleles were DRB1*16:01:01 (12.6%), DRB1*11:04:01 (12.1%) and DRB1*03:01:01 (12%). The HLA-DRB1*11:01:02 and -DRB1*08:04:01, -DRB1*05:01:01, -DRB1*13:05:01, -DRB1*14:07:01, -DRB1*09:01:02, -DRB1*11:02:01, -DRB1*04:07:01, -DRB1*15:03:01, -DRB1*03:02:01, -DRB1*04:06:02, -DRB1*04:08:01, -DRB1*14:05:01 were identified only once. The results revealed similarities with countries belonging to the Eastern Europe, the Balkans and the Caucasus regions. Further studies on larger Romanian cohorts are needed for confirming the current results.