Despite high morbidity and mortality due to Shigellosis especially in developing countries, unarguably due to poor personal hygiene and sanitary conditions.  There is dearth of literature on molecular characterization of Shigella species. In this prospective cross sectional study, Seven hundred diarrheal stool samples were analyzed. Participants consisted of 300 HIV positive and 400 HIV negative patients. The samples were consecutively collected from consenting patients that presented for care in the four main health facilities in Maiduguri, Borno state, northeastern Nigeria. Molecular detection of highly conserved 16s rRNA gene for identifying genetic properties were performed by polymerase chain reaction (PCR).  A total of 56 (8.0%) Shigella species, composed of 21 (7.0%) from HIV positive and 35 (8.7%) were isolated by conventional assay and 16s rRNA PCR. Molecular characterization of the 56 isolates based on more sensitive Multiplex PCR platform indicates only 32 isolates as Shigella species. They consisted of 14(45.2%) S. flexneri, 12(38.7%) S. dysenteriae and 5 (16.1%) S. sonnei. S. flexneri was commoner among HIV negative patients. There was no variation in the composition of the isolates based on gender or defined age group.