is a nontuberculous pathogen of poikilothermic fish and an opportunistic human pathogen. Like tuberculous mycobacteria, the M strain requires the ESX-1 (ESAT-6 system 1) secretion system for virulence in host cells. EsxB and EsxA, two major virulence factors exported by the ESX-1 system, are encoded by the genes within the ESX-1 locus. Deletion of the genes abrogates ESX-1 export and attenuates in and models of infection. Interestingly, there are several duplications of the and genes (, , , , and ) in the M genome located outside the ESX-1 locus. We sought to understand if this region, known as ESX-6, contributes to ESX-1-mediated virulence. We found that deletion of the _ gene alone or the entire ESX-6 locus did not impact ESX-1 export or function, supporting the idea that the genes present at the ESX-1 locus are the primary contributors to ESX-1-mediated virulence. Nevertheless, overexpression of the locus complemented ESX-1 function in the Δ strain, signifying that the two loci are functionally equivalent. Our findings raise questions about why duplicate versions of the genes are maintained in the M genome and how these proteins, which are functionally equivalent to virulence factors, contribute to mycobacterial biology. is the causative agent of the human disease tuberculosis (TB). There are 10.4 million cases and 1.7 million TB-associated deaths annually, making TB a leading cause of death globally. Nontuberculous mycobacteria (NTM) cause chronic human infections that are acquired from the environment. Despite differences in disease etiology, both tuberculous and NTM pathogens use the ESX-1 secretion system to cause disease. The nontubercular mycobacterial species, , has additional copies of specific ESX-1 genes. Our findings demonstrate that the duplicated genes do not contribute to virulence but can substitute for virulence factors in These findings suggest that the duplicated genes may play a specific role in NTM biology.