In order to study the genetic variability of Mycobacterium tuberculosis circulating in the Carabobo State at Venezuela, 317 strains of M. tuberculosis isolated from patients living in different health districts whose acid fast smears were positive, were included. Each strain was characterized by spoligotyping and the pattern compared with the national and worldwide SpolDB4.0 databases; 220 isolates (69.4%) were grouped in 24 clusters, being LAM the most common family (34%). In contrast to other Venezuelan regions, the most common Carabobo spoligotype was number 605, with 46 isolates (14.5%). In addition, there were 97 (30.6%) orphan spoligotypes, 19 of which are found in SpolDB4.0, and 78 non described. Co-infection with human immunodeficiency virus was detected in 11 patients (3.5%). These results show high genotypic variability of M. tuberculosis in the region, contributing with new information for a better understanding of tuberculosis transmission in Venezuela.