Background: Tuberculosis is the second largest public health threat caused by pathogens. Understanding Mycobacterium tuberculosis’s transmission, virulence, and resistance profile is crucial for outbreak control. This study aimed to investigate the pangenome composition of Mycobacterium tuberculosis clinical isolates classified as L4 derived from Ecuador. Methods: We analyzed 88 clinical isolates of Mycobacterium tuberculosis by whole-genome sequencing (WGS) and bioinformatic tools for Lineage, Drug-resistance and Pangenome analysis. Results: In our analysis, we identified the dominance of the LAM lineage (44.3%). The pangenomic analysis revealed a core genome of approximately 3200 genes and a pangenome that differed in accessory and unique genes. According to the COG database, metabolism-related genes were the most representative of all partitions. However, differences were found within all lineages analyzed in the metabolic pathways described by KEGG. Isolates from Ecuador showed variations in genomic regions associated with beta-lactamase susceptibility, potentially leading to epistatic resistance to other drugs commonly used in TB treatment, warranting further investigation. Conclusions: Our findings provide valuable insights into the genetic diversity of Mycobacterium tuberculosis populations in Ecuador. These insights may be associated with increasing adaptation within host heterogeneity, variable latency periods, and reduced host damage, collectively contributing to disease spread. The application of WGS is essential to elucidating the epidemiology of TB in the country.