Tuberculosis remains a challenge in both rural and urban areas. Although a majority of countries display a higher burden in urban areas compared with rural areas, Panama continues to report the highest mortality rate in Central America. Urban areas, such as Panama City, report a high tuberculosis burden, whereas Panama's western region, including the provinces of Chiriquí, Bocas del Toro (both semiurban) and Ngäbe-Bugle (rural), show a lower burden. We aimed to identify highly transmitted Mycobacterium tuberculosis strains within rural and semiurban settings of Panama's western region during a 3-year period (2017, 2019, 2021). We randomly selected 87 M. tuberculosis isolates from a biobank from Panama's western region and analyzed them using allele-specific oligonucleotide polymerase chain reaction and 24-mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR). Our results show only 11.7% (10/85) of M. tuberculosis strains identified as prevalent A-Beijing, B-Haarlem, or C-LAM Strains. We found a low prevalence of A, B, and C M. tuberculosis strains in both rural and semirural settings compared with isolates collected from the Eastern Colon Province. MIRU-VNTR genotyping revealed a high degree of diversity with no clusters with single loci variation of ≥ 2 loci. These results support the notion that tuberculosis prevalence in the rural and semiurban western region of Panama are not due to previously described highly transmitted strains but is influenced instead by other health determinants, including poor health system access and a lack of systematic transmission chain monitoring. For remote rural and semiurban settings, we recommend allocating resources to reinforce efforts to prevent tuberculosis spread.