Objective To understand the drug resistance of Mycobacterium tuberculosis (M. tuberculosis) in five drug resistance monitoring sites in Henan Province, and provide reference for formulating the control strategies of tuberculosis epidemic. Methods The strains and clinical information of 1 716 tuberculosis patients were collected from five drug resistance monitoring sites in Henan Province in 2013-2018. M. tuberculosis was tested for the sensitivity to nine anti-tuberculosis drugs by proportional method, changes in their drug resistance was analyzed. Results The total drug resistance rate and multidrug resistance (MDR) rate of tuberculosis patients were 24.07% (413/1 716) and 3.73% (64/1 716) respectively. Drug resistance rates of the first-line anti-tuberculosis drugs from high to low were as follows: streptomycin (SM) 13.34% (n=229), isoniazid (INH) 10.08% (n=173), rifampicin (RIF) 5.19% (n=89), and ethambutol (EMB) 4.49% (n=77). Drug resistance rates of the second-line anti-tuberculosis drugs from high to low were as follows: ofloxacin (OFX) 3.15% (n=54), kanamycin (KAM) 2.51% (n=43), capreomycin (CPM) 2.51% (n=43), p-aminosalicylic acid (PAS) 1.98% (n=34), and prothionamide (PTO) 1.46% (n=25). Drug resistance profile showed that 413 strains were distributed among 68 different drug resistance types, mono-drug resistance rate of SM, poly-drug resistance of INH+SM, as well as MDR rates of INH+RIF and INH+RIF+SM were higher. The drug resistance rates and MDR rates of initial treated patients were 20.13% (216/1 073) and 2.80% (30/1 073), respectively. Drug resistance rates and MDR rates of retreated patients were 30.64% (197/643) and 5.29% (34/643), respectively. Drug resistance rates and MDR rates of retreated patients were both higher than those of initial treated patients (both P < 0.05). Patients aged 41 to 60 years had the highest drug resistance rate of 29.25%, and patients aged 2 to 20 years had the lowest drug resistance rate of 13.56%. There was statistically significant difference in drug resistance rates among different age groups (P < 0.05). Drug resistance trend analysis showed that only the MDR rate increased year by year in 2013-2018 (P < 0.05). Conclusion Drug resistance of Mycobacterium tuberculosis in five drug resistance monitoring sites in Henan Province is still serious, drug resistance profile showed that the types of drug resistance were diverse and complicated, drug resistance rates of patients aged 41-60 years and retreated patients are high, and MDR rate showed a rising trend. Therefore, it is necessary to standardize treatment, carry out screening on drug resistance, reduce the occurrence of drug-resistant patients, strengthen the treatment and management of MDR patients to prevent transmission.