Tuberculosis (TB) is an airborne infectious disease caused by Mycobacterium tuberculosis with a global health impact of 1.3 million deaths and 7.5 million cases in 2022. It is highly prevalent in countries of Sub-Saharan Africa and South Asia. In Oman, TB is of low burden with an incidence rate of 9.3 cases per 105 population in 2022. The disease is curable; however, treatment is challenged by the emergence of drug resistance. Drug resistant TB in Oman is limited; however, recent studies revealed an increase in pyrazinamide resistance (PZAR). Thus, the present study aims to determine the extent of genetic diversity among PZAR isolates and define mutations in three candidate genes associated with phenotypic resistance to the drug. A total of 179 Mtb isolates, comprising PZAR (n=105) and PZAS (n=74), were obtained from the Central Public Health Laboratories, Ministry of Health, Oman, between 2019 and 2023, from Omanis (n=56) and expatriates (n=123). Genetic diversity was determined by spoligotyping, and three genes implicated in PZA resistance (pncA, panD, and rpsA) were analyzed by Sanger sequencing. To assess evolution of PZAR strains, Mtb isolates examined in the current study were compared to those collected between 2014 and 2018. High diversity, comprising 10 spoligoclades, was seen among the 2019-2023 Mtb isolates. The most prevalent spoligoclades were EAI (57.5%), CAS (15.8%), and TH (9.6%). Low diversity was seen among PZAR (2 clades) compared to PZAS isolates (10 clades). No significant differences existed between the spoligoprofile of PZAR strains among Omanis and expatriates. The spoligoprofile of the Mtb isolates examined in the current study differed from that seen in an earlier population (2014-2018). The major spoligoclades (CAS, EAI, T, and orphan) existed at significantly different frequencies in the two populations. Similarly, significant differences were seen in the spoligoprofile of PZAR isolates examined here compared to those of 2014-2018. Low frequency of mutations was seen among pncA, panD, and rpsA. This included, 6 pncA mutations, comprising 1 indel, 2 nonsynonymous, and 3 synonymous mutations. Similarly, 5 panD mutations were identified, 4 nonsynonymous and 1 synonymous, and 4 rpsA mutations consisting of 2 nonsynonymous and 2 synonymous were determined. The majority of the mutations in pncA, panD, and rpsA were found in the EAI spoligolineage, followed by CAS and Beijing. With PZAS as the reference, single-gene pncA, panD, and rpsA sequencing had a sensitivity of 3.1%, 1.1%, 4.7% and a specificity of 100.0%, 95.5%, 100.0%, respectively. However, in combination, the three genes increased the sensitivity to 7.8% and resulted in a specificity of 97.5%. In summary, PZAR Mtb strains in Oman showed limited diversity, with PZAR conferring mutations were clustered in the EAI spoligoclade, indicative of a limited resistance reservoir. pncA, panD, and rpsA have limited diagnostic sensitivity to PZAR Mtb strains in Oman, suggesting the involvement of other genes.