Thailand has a high burden of tuberculosis, with control efforts hindered by drug-resistant Mycobacterium tuberculosis (Mtb). The increasing use of whole-genome sequencing (WGS) of Mtb offers valuable insights for clinical management and public health surveillance. WGS can be used to profile drug resistance, identify circulating sub-lineages, and trace transmission pathways or outbreaks. We analysed WGS data from 2,005 Mtb isolates collected across Thailand from 1994-2020, including 816 retrieved and 1,189 newly sequenced samples, with most isolates being multidrug-resistant (MDR-TB). Most isolates are lineage two strains (78·3%), primarily the Beijing sub-lineage (L2.2.1). Drug resistance profiling revealed substantial isoniazid and rifampicin resistance, and 67·3% classified as MDR-TB. Phenotypic and genotypic drug susceptibility testing showed high concordance (91·1%). Clustering analysis identified 206 transmission clades (maximum size 288), predominantly with MDR-TB, especially in Central and Northeastern regions. One cluster (n = 22) contains the ddn Gly81Ser mutation, linked to delamanid resistance, with some members pre-dating drug roll-out. In the largest cluster (n = 288), containing isolates spanning two decades, we applied transmission reconstruction methods to estimate a mutation rate of 1·1 × 10 substitutions per site per year. Overall, this study demonstrates the value of WGS in uncovering TB transmission and drug resistance, offering key data to inform better control strategies in Thailand and elsewhere.