This paper evaluates the capability of MicroSeq 500 instrument to improve the diagnosis of Mycobacterium genavense. The strain was isolated from a two year old child admitted to our hospital for hepatosplenomegaly and massive abdominal lymphadenopathies. DNA was extracted from a lymph node and examined by amplifying 500 bp at the 5′ end of 16S rRNA gene using MicroSeq 500 16S rDNA Bacterial Identification PCR kit. Sequencing reactions were performed with MicroSeq 500 16S rDNA Bacterial Identification Sequencing kit (Applied Biosystems, USA). Afterwards, sequences were analyzed by GenBank database and identified as Mycobacterium genavense, a slow-growing nontuberculous mycobacterium. The use of 16S rRNA gene sequencing for the identification of bacteria allows the recognition of new clinically relevant agents, eliminating the culture result waiting times.