Bacillus thuringiensis was isolated from 116 samples collected in high altitude potato-growing areas in Bolivia. In these regions, main potato pests are the potato tuberworm Phthorimaea operculella, and the Andean weevils Premnotrypes latithorax and Rhigopsidius tucumanus. B. thuringiensis was found in 60% of the samples. The main percentage of samples with B. thuringiensis was found in larvae of R. tucumanus (78%). Bioassays were performed with 112 isolates. None resulted toxic to either larvae or adults of the two Andean weevils. However, 18 isolates from this study showed more toxicity against the beet armyworm Spodoptera exigua than the standard strain var. kurstaki isolated from DELFIN. Among these isolates, three were also effective against P. operculella, conferring better or equal protection to the tubers than the reference strain HD-1 isolated from DIPEL. The most toxic strains against S. exigua and P. operculella were characterized in terms of serotyping, crystal morphology, protein profile, and cry gene content. PCR was performed with primers amplifying genes from the cry1, cry2, cry3, cry4, cry7, 8, and cry9Aa families. The toxic strains presented bipyramidal crystals, at least a band of 130kDa in SDS-PAGE, and showed an amplification product with cry1 family primers. One of the isolates did not amplify with any specific primer belonging to known cry1 genes. Restriction Fragment Length Polymorphism (RFLP) confirmed the presence of a novel gene and sequence comparison showed that this gene had homology to cry1G.