The objectives of this work were to evaluate the genetic variability of Meloidogyne enterolobii by molecular markers, and develop species-specific molecular markers for application in detection. Sixteen M. enterolobii isolates from different geographical regions (Brazil and other countries) and hosts were used in this study. The identification and purification of the populations were carried out based on isoenzyme phenotype. The DNA amplification of the intergenic region (IGS) of the rDNA and of the region between the cytochrome oxidase subunit II (COII) and 16S rRNA genes (mtDNA) produced specific fragments of the expected size for this nematode, i.e. 780 and 705 bp, respectively. Intraspecific variability among the isolates was evaluated with three different neutral molecular markers: AFLP, ISSR and RAPD. The results showed a low level of diversity among the isolates tested, indicating that M. enterolobii is a genetically homogeneous root-knot nematode species. The RAPD method allowed the identification of a species-specific RAPD fragment for M. enterolobii. This fragment was cloned and sequenced, and from the sequence obtained, a set of primers was designed and tested. The amplification of a 520-bp-long fragment occurred only for the 16 isolates of M. enterolobii and not for the 10 other Meloidogyne species tested. In addition, positive detection was achieved in a single individual female, egg-mass and second stage juvenile of this nematode. This SCAR species-specific marker for M. enterolobii represents a new molecular tool to be used in the detection of this nematode from field samples and as a routine diagnostic test for quarantine devices.