Four collections comprising 507 strains of Bacillus thuringiensis have been analysed in this study. A different ecological origin characterizes each collection. Collection No. 1 was established from soil, dust, and grain samples from Spanish agricultural and non-cultivated soil, silos, and mills. Collection No. 2 is the result of a screening in olive-crop related environments in Spain. Collection No. 3 is made up of strains isolated from potato-growing areas in Bolivia. Collection No. 4 has been generated for this study and includes strains collected from diverse types of samples belonging to several habitats from Spain and Mexico. Crystal morphologies and cry1A and cry2 gene content were assessed for all isolates from each collection. In the 507 strains, the most common crystal morphology was bipyramidal crystals. Frequencies of cry1A and cry2 genes were 61.5% and 59.2%, respectively, and there was a strong correlation between the occurrence of cry1A and cry2 genes.