Bacillus thuringiensis is a Gram-positive, aerobic, facultative anaerobic and endosporeforming bacterium. Different strains of this species have the ability to produce parasporal crystalline inclusions which are toxic to larvae of different insect orders and other invertebrates and cause rapid death of the host. To determine the importance of this species in microbial control, we collected native strains and studied their virulence on the diamondback moth, Plutella xylostella. More than 148 samples were collected from Alborz, Guilan and Mazandaran Provinces. Experimental samples, including soil samples from forests, fruit gardens, agricultural fields, diseased and dead larvae, were transferred to a laboratory in sterile plastic containers. For evaluating B. thuringiensis isolates virulence, a cabbage leaf dip method with 106 cell ⋅ ml–1 concentration of various Bt isolates was applied to diamondback moths. Larval mortality was recorded 72 h after treatment. Based on bioassay results, all isolates were classified into three high, medium and low virulence groups. Protein level characterization based on the SDS-PAGE gel analysis showed that two isolates from a high virulence group have proteins of high molecular masses of 121 and 109 kDa. Results revealed that there is a positive correlation between protein masses and virulence of isolates. In addition, this research introduced nine strains that are highly toxic to P. xylostella and would be valuable as insecticidal agents for controlling lepidopteran pests.