A total of 27 entomopathogenic nematode (EPN) strains originally isolated from different cotton fields were characterized in laboratory experiments for their virulence, reproductive potential and environmental tolerance. The EPN strain collections included 16 Steinernema carpocapsae (SC), three Steinernema siamkayai (SS), one Steinernema monticolum (SM), and seven Heterorhabditis bacteriophora (HB). Their virulence was tested against cotton bollworms such as the American bollworm Helicoverpa armigera, the spotted bollworm Earias vittella and the cotton leafworm Spodoptera litura. Larvae of H. armigera, E. vittella and S. litura were susceptible to all the tested EPN species/strains with significant differences among EPN species/strains. The most virulent strains were APKS2 (SC), TRYH1 (HB) KKMH1 (HB) and KKMH2 (HB) on H. armigera (91.9–93.5% mortality); KKMS1 (SC), APKS2 (SC), TRYH1 (HB), KKMH1 (HB), KKMH2 (HB) and APKH1 (HB) on E. vittella (92.7% mortality); and APKS2, TRYH1, KKMH1, KKMH2 and KKMH3 on S. litura (92.7% mortality). The results of the invasion rate assay indicated that the EPN strains more virulent against the target host had greater invasion rates. In the multiplication assay, KKMH1and OCMS1 (SC) produced a greater number of infective juveniles (IJs) (32.1–32.4 x 1000 IJs/ cadaver) in Carcyra cephalonica. Test for tolerance to heat at 40°C for 2 h revealed that KKMH1, TRYH1, KKMH2, KKMS1 and APKS2 were highly tolerant (>85% survival). IJs exposed to ambient room conditions (27–29°C; 65–70% r.h.) for 2 h showed that APKS2, OCMS1 and KKMS1 were more tolerant (68–69% survival) of rapid desiccation than others. APKS2, KKMS1 and KKMH1 showed better survival (70–73%) in slow desiccation assay when exposed to 25°C with 97% r.h. for 72 h, followed by 25°C with 93% r.h. for 48 h. The H. bacteriophora KKMH1 and S. carpocapsae APKS2 performed best in nine traits out of ten tested, followed by H. bacteriophora TRYH1, which performed best for six traits. It is suggested that the EPN strains KKMH1 and APKS2 could be deployed for a cotton bollworm management program after testing their performance under field conditions.