Although in Bacillus thuringiensis the cry genes coding for the insecticidal crystal proteins are plasmid-borne and are usually associated with mobile genetic elements, several aspects related to their genomic organization, diversification, and transmission remain to be elucidated. Plasmids of B. thuringiensis and other members of the Bacillus cereus group (n = 364) deposited in GenBank were screened for the presence of cry1 genes, and their genetic environment was analyzed using a comparative bioinformatic approach. The cry1 genes were identified in 27 B. thuringiensis plasmids ranging from 64 to 761 kb, and were predominantly associated with the ori44, ori60, or double orf156/orf157 and pXO1-16/pXO1-14 replication systems. In general, the cry1 genes occur individually or as a part of an insecticidal pathogenicity island (PAI), and are preceded by genes coding for an N-acetylmuramoyl-l-alanine amidase and a putative K+(Na+)/H+ antiporter. However, except in the case of the PAI, the latter gene is disrupted by the insertion of IS231B. Similarly, numerous mobile elements were recognized in the region downstream of cry1, except for cry1I that follows cry1A in the PAI. Therefore, the cassette involving cry1 and these two genes, flanked by transposable elements, named as the cry1 cassette, was the smallest cry1-carrying genetic unit recognized in the plasmids. Conservation of the genomic environment of the cry1 genes carried by various plasmids strongly suggests a common origin, possibly from an insecticidal PAI carried by B. thuringiensis megaplasmids.