This study investigates the genetic structure and demographic history of contemporary Thai populations from Tak and Ranong, two border provinces between Thailand and Myanmar with complex immigration histories. We analyzed 20 Y-SNP markers (327 samples) and 24 Y-STR profiles (241 samples), along with published datasets, and observed significant genetic differentiation between these regions. Tak is dominated by O2a2b1a1a (44.53%) and O1b1a1a (21.17%). The presence of NO1 (14.6%) suggests genetic contributions from nearby populations such as the Karen, Lawa, and Khuen. Notably, Tak shows low gene diversity (GD) at the DYS391 (0.2208), contrasting with Ranong (0.5247) and the overall Thailand population (0.4708). Ranong exhibits a more diverse haplogroup distribution, with prominent frequencies of O1b1a1a (20.75%), F (16.98%) and O2a2b1a1a (13.21%). The presence of haplogroups K (11.32%) and R* (7.55%) reflects a genetic connection with the Maniq or other mainland Negrito groups, as well as historical gene flow from South Asia. These findings enhance our understanding of the genetic landscape shaped by migration and localized admixture. We also expanded the Thailand Y-chromosome database with novel Y-STR haplotypes, updated forensic parameters and Y-haplogroup frequencies, providing valuable resources for paternal ancestry inference and kinship verification in both forensic science and population genetics.