Citrus tristeza virus (CTV) is an important issue to the Mexican citrus industry. CTV infected plants have been reported in 20 citrus producing states of the country as symptomless virus infections, even on plants grafted on the sensitive sour orange rootstock. The detection of severe CTV isolates is mandatory to reinforce the operation of the CTV campaign nationwide. In this study, 13 CTV isolates from Nuevo Leon and one from Tamaulipas states were characterized for activity against the strain discriminatory MCA13 monoclonal antibody, bi-directional (BD) PCR, and hybridization with specific DNA probes, with RT-PCR amplicons, of the p25 coat protein gene. Ten out of 13 CTV isolates from Nuevo Leon and the Tamaulipas isolate gave a positive reaction to the MCA13 monoclonal antibody. BD-PCR tests yielded a cDNA fragment of 300 bp characteristic of decline-inducing CTV strains with eight of 13 isolates from Nuevo Leon and the Tamaulipas isolate. Hybridization with strain group specific DNA probes showed reactivity of eight CTV isolates with Probe II associated with decline inducing CTV strains, and three CTV isolates with Probe VII, associated to mild CTV strains. As a conclusion, it was shown the usefulness of the MCA13 monoclonal antibodies, BD-PCR and the hybridization with strain group specific DNA probes (SGSP) for the discrimination of CTV isolates, and the discovering of severe CTV isolates in the state of Nuevo Leon. Both BD-PCR and hybridization with SGSP were more suitable for strain discrimination of CTV isolates, than the MCA13 monoclonal antibody. BD-PCR was able to detect mixtures of both mild and decline inducing CTV isolates in the sample; hybridization with SGSP was suitable for detecting either mild, decline inducing and stem pitting CTV isolates.