Objective To analyze the infection trend, distribution characteristics and epidemic status of non-tuberculous mycobacterial (NTM) infection in Yichang area by fluorescence polymerase chain reaction (PCR) melting curve method, providing reference for the prevention and treatment of NTM infection in this area. Methods Pleural effusion, peritoneal effusion, sputum and bronchoalveolar lavage fluid from patients with suspected tuberculosis in a hospital in Yichang area from 2016 to 2020 were collected. All Mycobacterium positive specimens detected by Ziehl-Neelsen acid-fast staining smear, MGIT 960 rapid culture and modified Loewenstein-Jenson medium culture were further identified Mycobacterium tuberculosis (MTB) and NTM by fluorescence PCR melting curve method. With whole genome sequencing (WGS) as the gold standard, the accuracy of fluorescence PCR melting curve method in identifying MTB and NTM was compared and analyzed, and clinical characteristics of NTM infected patients in Yichang area were summarized. Results A total of 7 496 Mycobacterium positive specimens were collected. After identified by fluorescence PCR melting curve method, 6 997 (93.34%) MTB strains and 499 (6.66%) NTM strains were detected. A total of 469 (93.99%) NTM strains were identified, while the remaining 30 (6.01%) strains were unknown strains. The most commonly detected NTM was Mycobacterium intracellulare (n=189, 37.88%), followed by Mycobacterium abscessus (n=68, 13.63%) and Mycobacterium gordonae (n=25, 5.01%). 22 MTB strains and 18 NTM strains identified by fluorescence PCR melting curve method were selected to perform WGS, and the coincidence rate, sensitivity, and specificity were 98.15%, 99.80%, and 93.54%, respectively. The epidemic characteristics of NTM showed that the detection rate of NTM in this area has been increasing year by year, from 3.56% in 2016 to 13.21% in 2020. Among NTM positive patients, 57.52% (287/499) were male, 52.30% (261/499) of the patients were aged 55-74 years old. Conclusion The prevalence of NTM in Yichang area presents a rapid increasing trend, with Mycobacterium abscessus, Mycobacterium gordonae and Mycobacterium kansasii as the main pathogens. Fluorescence PCR melting curve method can effectively and quickly assist in diagnosis. The risk of NTM infection in elderly people is high, and differential diagnosis is of great significance in clinical practice.