A Gram-positive, acid-fast and rapidly growing rod, designated S2-37 ^T, that could form yellowish colonies was isolated from one soil sample collected from cotton cropping field located in the Xinjiang region of China. Genomic analyses indicated that strain S2-37 ^T harbored T7SS secretion system and was very likely able to produce mycolic acid, which were typical features of pathogenetic mycobacterial species. 16S rRNA-directed phylogenetic analysis referred that strain S2-37 ^T was closely related to bacterial species belonging to the genus Mycolicibacterium, which was further confirmed by pan-genome phylogenetic analysis. Digital DNA-DNA hybridization and the average nucleotide identity presented that strain S2-37 ^T displayed the highest values of 39.1% (35.7–42.6%) and 81.28% with M. litorale CGMCC 4.5724 ^T, respectively. And characterization of conserved molecular signatures further supported the taxonomic position of strain S2-37 ^T belonging to the genus Mycolicibacterium. The main fatty acids were identified as C_16:0, C_18:0, C_20:3ω3 and C_22:6ω3. In addition, polar lipids profile was mainly composed of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. Phylogenetic analyses, distinct fatty aids and antimicrobial resistance profiles indicated that strain S2-37 ^T represented genetically and phenotypically distinct from its closest phylogenetic neighbour, M. litorale CGMCC 4.5724 ^T. Here, we propose a novel species of the genus Mycolicibacterium: Mycolicibacterium gossypii sp. nov. with the type strain S2-37 ^T (= JCM 34327 ^T = CGMCC 1.18817 ^T).