Investigating genetic structure and diversity is crucial for the rye hybrid breeding strategy, leading to improved plant productivity and adaptation. The present study elucidated the population structure and genetic diversity of 188 rye accessions, comprising 94 pollen fertility restoration lines (RF) and 94 cytoplasmic male-sterile (CMS) lines with Pampa sterilizing cytoplasm using SNP and silicoDArT markers from the diversity array technology (DArT)-based sequencing platform (DArTseq). Expected heterozygosity ( H e ) and Shanon’s diversity ( I) indexes varied slightly between marker systems and groups of germplasms ( H e = 0.34, I = 0.51 for RF and CMS lines genotyped using SNPs; H e = 0.31, I = 0.48, and H e = 0.35, I = 0.53 for RF and CMS using silicoDArTs, respectively). ANOVA indicated moderate variation (7%) between RF and CMS breeding materials. The same parameter varied when chromosome-assigned markers were used and ranged from 5.8% for 5R to 7.4% for 4R. However, when silicoDArT markers were applied, the respective values varied from 6.4% (1R) to 8.2% (3R and 4R). The model-based (Bayesian) population structure analysis based on the total marker pool identified two major subpopulations for the studied rye germplasm. The first one (P1) encompasses 93 RF accessions, and the second one (P2) encompasses 94 CMS and one RF accession. However, a similar analysis related to markers assigned to selected chromosomes failed to put plant materials into any of the populations in the same way as the total marker pool. Furthermore, the differences in grouping depended on marker types used for analysis.