Nontuberculous mycobacteria (NTM) is a large group of mycobacteria other than the Mycobacterium tuberculosis complex and Mycobacterium leprae. Epidemiological investigations have found that the incidence of NTM infections is increasing in China, and it is naturally resistant to many antibiotics. Therefore, studies of NTM species in clinical isolates are useful for understanding the epidemiology of NTM infections. The present study aimed to investigate the incidence of NTM infections and types of NTM species. Of the 420 samples collected, 285 were positive for M. tuberculosis, 62 samples were negative, and the remaining 73 samples contained NTM, including 35 (8.3%) only NTM and 38 (9%) mixed (M. tuberculosis and NTM). The most prevalent NTM species were Mycobacterium intracellulare (30.1%), followed by Mycobacterium abscessus (15%) and M. triviale (12%). M. gordonae infection was detected in 9.5% of total NTM-positive cases. Moreover, this study reports the presence of Mycobacterium nonchromogenicum infection and a high prevalence of M. triviale for the first time in Henan. M. intracellulare is the most prevalent, accompanied by some emerging NTM species, including M. nonchromogenicum and a high prevalence of M. triviale in Henan Province. Monitoring NTM transmission and epidemiology could enhance mycobacteriosis management in future.