Coix lacryma-jobi L. is a cereal crop belonging to the Poaceae family, valued for its nutritional, medicinal, and ornamental uses. Among its varieties, two prominent landraces, C. lacryma-jobi var. kiboa and var. tapol, are cultivated for their distinct grain morphologies and favorable agronomic traits. However, despite its economic importance, detailed cytogenetic and molecular phylogenetic studies remain limited. This study presents a cytogenetic and molecular phylogenetic analysis of C. lacryma-jobi L. var. kiboa and tapol. Partial cytogenomic characterization on the 45S and 5S rDNA loci as well as Arabidopsis- type telomeric repeats using pre-labeled oligomer probes for fluorescence in situ hybridization (PLOP-FISH) technique confirmed a diploid chromosome number of 2n = 20 in both varieties. The 45S and 5S rDNA loci were localized on the nucleolar organizer region (NOR) of the short arm of chromosome 1 and the long arm of chromosome 4, respectively, indicating conserved chromosomal arrangements. Additionally, Arabidopsis-type telomeric repeats were detected at the terminal regions of all chromosomes. Chromosome sizes ranged from 2.98 ± 0.07 to 3.74 ± 0.11 μm in var. kiboa and from 3.42 ± 0.08 to 4.12 ± 0.05 μm in var. tapol. Phylogenetic analyses based on the internal transcribed spacer (ITS1 and ITS4) genes revealed a close genetic relationship between the two varieties, supporting their shared evolutionary lineage. These findings enhanced our understanding of adlay genetic diversity and provide foundational insights for plant breeding improvement, conservation strategies, and future genomic research.