Our study surveyed Meloidogyne spp. from tobacco cultivars in Brazil carrying the Rk gene, which confers resistance to Meloidogyne incognita races 1 and 3. Identification of Meloidogyne populations from 39 tobacco fields was based on the analysis of esterase phenotypes, perineal patterns, and 18S-internal transcribed spacer (ITS)1-5.8S ribosomal RNA sequences. Biometric characterization and differential host tests of isolates were determined as well. We detected M. incognita, M. javanica, M. enterolobii, M. arenaria, and M. inornata in 18 (46.2%), 16 (41%), 10 (25.6%), 2 (5.1%), and 1 (2.6%) samples, respectively. Mixtures of species were found in 25.6% of the samples. This is the first report of M. inornata parasitizing tobacco in Brazil’s southern region. Two morphological and biochemically unusual populations had host ranges and ITS1 sequences nearly identical to M. enterolobii and M. incognita, respectively. Pathogenic and biometric intraspecific variations were observed. Based on our results and considering the limited efficiency of the Rk gene, it is indispensable not only to search for resistance sources to M. incognita virulent strains but also to consider resistance to M. enterolobii, M. inornata, M. arenaria, and M. javanica in tobacco breeding programs. Changes in integrated management procedures should be considered to avoid increased crop damage in the future.