Mycobacterium tuberculosis lineage 2 (L2) remains a globally significant lineage associated with increased drug resistance and rapid transmission. The L2 lineage exhibits a hotspot for genetic diversity and evolution in Panama, requiring an in-depth analysis. We conducted a prospective analysis of 274 Mycobacterium tuberculosis L2 isolates from Colon City between January 2021 and October 2023. Drug resistance was determined using GeneXpert and MTBDRplus-Genotype assays, strain lineage was determined by strain-specific PCR (ASO-PCR), and whole-genome sequencing was conducted for phylogenetic analysis. Sequencing data were analyzed using the mtb-call2 pipeline and TB-gen tools to predict drug resistance and sublineage, respectively. Genome-wide single-nucleotide polymorphisms (SNPs) were used for phylogenetic and evolutionary analyses. ASO-PCR results identified all 31.7 % (86/271) isolates as Modern L2.2. WGS analysis of 66 strains confirmed all isolates belonged to the L2.2.1 sublineage. Sixty-four strains were analyzed in depth, with 96.9 % (62/64) classified as pan-susceptible and 3.1 % (2/64) as rifampicin/pyrazinamide-resistant. The sublineage analysis based on SNPs using the TB-gen tool identified a SNP at position 1219683G >
A, which genotyped all 64 strains as L2.2.M3 sublineage. Phylogenetic analysis revealed a correlation with geographical distribution compared to other Latin American L2 isolates. Transmission clusters (≤12 SNPs) were identified and used to determine recent transmission events or TB transmission clusters. These analyses also confirmed a relatively low evolutionary rate within Panama L2 isolates and a highly conserved common ancestor shared with L2 isolates from Peru, Colombia, and Guatemala. These findings suggest endemic transmission of the Mycobacterium tuberculosis L2.2.M3 sublineage in Colon, Panama. We recommend combining genomic information with epidemiological data to accurately track and identify the source hotspot for the L2.2.M3 sublineage and focus control measures.