Objective To understand the genotypes and drug resistance of Mycobacterium avium (MAV) from persons with HIV/MAV double infection in southwest region of Yunnan Province. Methods 12 specific loci of variable-number tandem repeat (VNTR) were chosen, and genotyping of MAV was performed with MAV tandem repeats (MATR-VNTR). DNA of the strains was amplified by polymerase chain reaction (PCR), and DNA fingerprint of MAV was obtained by agarose gel electrophoresis. The electrophoresis result was digitized by Quantity One and BioNumerics 6.7 software, and cluster analysis was carried out by unweighted pair-group method with arithmetic means (UPGMA). Susceptibility of 44 MAV strains to 10 different antimicrobial drugs was tested by proportional test method. Results The Hunter-Gaston discriminatory index (HGDI) of VNTR genotyping of each locus was different. MATR-5 was the highest (0.68). HGDI values of MATR-1, 2, 3, 4, 6, 7, 9, 13, and 15 were all ≥ 0.5. Through cluster analysis, 44 MAV strains were divided into 6 clusters. Antimicrobial susceptibility results showed that rifabutin had the best in vitro antimicrobial activity against MAV (resistance rate 0), followed by ethambutol (resistance rate 86.4%, 38/44). All strains were resistant to the other 8 antimicrobial agents. Conclusion VNTR genotyping method of the selected 12 loci has high resolution in southwest Yunnan, and antimicrobial resistance of MAV strains from different regions in southwest region of Yunnan Province is serious.