Risk factors of isoniazid mono-resistance were reported by N-acetyltransferase 2 (NAT2) gene and some clinical factors in the literature. This study aimed to determine the risk factors of isoniazid mono-resistance among Thai pulmonary tuberculosis patients. The case-control study was conducted from 18th September 2017 to 20th February 2018. Demographic data were collected from medical record review and interview. NAT2 genotypes were collected from saliva samples collection and determined by multiplex polymerase chain reaction and gel electropherosis. Risk factors were conducted by multivariate logistic regression analysis and strength of association was reported as odd ratio with 95% confident interval. Total 167 participants comprised of 50 isoniazid mono-resistant and 117 drug-susceptible tuberculosis patients. NAT2*4/*6A was the most common genotype in both 12 (24.0%) cases and 38 (32.5%) controls, respectively, and there was not significantly different among genotypes between two groups (p-value = 0.352). Moreover, rapid acetylator was not significantly associated with isoniazid mono-resistance compared with slow acetylator (OR 1.169; 95%CI 0.435-3.140, p-value = 0.389). Adults who were less than 50 years old were more likely to develop isoniazid mono-resistance (adjusted OR 2.281; 95%CI 1.101-4.728, p-value = 0.027) than elderlys who were older than 50 years old. In contrast, ever-drinkers were less likely to develop isoniazid mono-resistance (adjusted OR 0.417; 95%CI 0.207-0.842, p-value = 0.015). In conclusion, the young adults and never-drinkers are risk factors of isoniazid mono-resistance in these Thai pulmonary tuberculosis patients. Moreover, a further study is needed to clarify the role of NAT2 on isoniazid mono-resistance in Thais.