doi:10.1007/s10482-026-02301-1engTristancho Baró, AlexanderMilagro, AnaLópez-Calleja, Ana IsabelBadenas-Alzugaray, DavidBurillo, NataliaSanz, SaraGuardingo, MartaMartínez, NievesSamper, SofíaGarcía, María JesúsRezusta, AntonioViñuelas Bayón, Jesús MaríaMycobacterium cajalii sp. nov., a novel scotochromogenic rapid-growing nontuberculous mycobacterial species closely related to Mycobacterium servetiiART-2026-148906Non-tuberculous mycobacteria (NTM) represent an expanding group of environmental microorganisms with growing clinical significance, particularly among immunocompromised patients. In this study, we describe the isolation and comprehensive characterization of a novel Mycobacterium species, strain HUMS_1102779-3, recovered from a sputum sample of a patient undergoing diagnostic bronchoscopy for hilar lymphadenopathy. Initial identification attempts using MALDI-TOF MS and commercial DNA∙STRIP assays were inconclusive, prompting whole-genome sequencing for comprehensive genomic and phenotypic characterization. De novo genome assembly resulted in a 6.2 Mb draft genome with a GC content of 69.03 mol% and high completeness (98.4% conserved orthologues). Phylogenetic and genomic comparisons suggest close relationship to Mycobacterium servetii (ANI 94.52%, dDDH 63.5%), yet below the accepted species delineation thresholds (95% and 70%, respectively), thereby supporting its status as a distinct taxon within the Mycobacterium simiae complex. Phylogenomic reconstruction placed HUMS_1102779-3 within a monophyletic cluster neighboring M. servetii and Mycobacterium helveticum, but genetically divergent from them. The genome encoded 5833 coding sequences, including determinants associated with antimicrobial resistance (rbpA, rpoB-like variant, mtrA, efpA), as well as virulence-related genes typical of mycobacteria, such as components of the Type VII secretion system and iron-regulated factors (ideR, hbhA). No mobile genetic elements were detected. Phenotypically, the isolate is a fast-growing, scotochromogenic, acid-fast bacillus that exhibits notable dimorphism on 7H11 agar and produce rough yellow–orange colonies after 4–5 days at 35 °C. It differs from M. servetii by several biochemical traits (catalase negative, Tween 80 hydrolysis positive, tellurite reduction-positive) and distinct antimicrobial susceptibility patterns, notably higher MICs for linezolid and minocycline and lower for ciprofloxacin. Based on genomic, phylogenetic, and phenotypic evidence, we propose the designation Mycobacterium cajalii sp. nov.,. The type strain is HUMS_1102779-3 (= CECT 31296 = DSMZ 120455), with the genome deposited under genome assembly accession number ASM4580900v1.2026http://zaguan.unizar.es/record/17043510.1007/s10482-026-02301-1http://zaguan.unizar.es/record/170435oai:zaguan.unizar.es:170435ANTONIE VAN LEEUWENHOEK INTERNATIONAL JOURNAL OF GENERAL AND MOLECULAR MICROBIOLOGY 119, 92 (2026), 14byhttps://creativecommons.org/licenses/by/4.0/deed.esinfo:eu-repo/semantics/openAccess