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The forage cactus plant belongs to the family Cactaceae, genera Opuntia and Nopalea. Its origin and dispersion is located in Mexico - a region where great genetic diversity at inter- and intra-specific levels exists. In Brazil, this crop has great economic interest because such plants are the nutritional base for livestock from semi-arid regions due to the large amounts of water which they retain. However, little is known of endophytic diazotrophs in this cultivation. This work is aimed at characterizing molecularly 22 isolates from 7 cultivars of forage cactus plants (Opuntia spp.) - 'IPA 90-92'; 'Marmillon Fodder' (1317); 'F8' (438); 'F3 Rojo Vigor'; 'Mexico vegetable' (1294); 'Chile Fruit' (1371); 'COPENA -V1' - isolated in different semi-solid media specific for diazotrophic bacteria. Analyses were conducted using the following molecular markers: restriction techniques through endonucleases from gene 16S ribosomal rDNA (ARDRA - Amplified Ribosomal DNA Restriction Analysis) and ISSR microsatellites (Inter Simple Sequence Repeat). Of the 22 isolates, 8 were grown in NFb medium in the following cultivars of cactus pear: 'Marmillon Fodder' (1317), 'F8' (438) and 'Mexico vegetable' (1371). Two isolates (IPA-IS6 and IPA-IS8) were grown in JNFb medium in the cultivars 'Marmillon Fodder' and 'F3 Rojo Vigor'. The three isolates IPA-IS2, IPA-IS13 and IPA-IS15 were grown in JNFb medium in 'Marmillon Fodder', 'F3 Rojo Vigor' and 'IPA 90-92'. Six isolates were grown in LGI-P medium (Gluconacetobacter diazotrophicus) in the forage cactus 'Marmillon Fodder', 'F3 Rojo Vigor', 'F8' (438), 'Chile Fruit' (1371) and 'IPA 90-92'. And three isolates in the LGI medium -IPA-IS20 ('IPA 90-92'), IPA-IS17 ('COPENA-V1') and IPA-IS9 ('Chile Fruit' 1371). The ARDRA technique showed lower polymorphism of the ISSR technique and the use of more than one molecular technique may be ideal for a study of diversity in microorganisms. This research points to great diversity of bacteria living in association with cactus and it will be necessary to undertake more detailed studies with molecular markers to obtain a conclusive characterization of the genetic diversity of plants with these endophytes, which could be achieved working with ribosomal genes and subsequent sequencing of these microorganisms.