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The efficacy of the newly developed anticoagulant rodenticide, difethialone was evaluated against various rodent species in laboratory and fields. Difethialone at 0.025% concentration in the form of loose bait (broken wheat and rice + vegetable oil + garlic powder) gave absolute mortality in Rattus rattus, Mus musculus, Funambulus pennanti, and Meriones hurrianae during ‘no-choice’ tests in one day feeding. No significant difference was noted in poison bait intake and mean days to death between two and three days poison feeding. Mean days to death were ranged between 2.9 to 5.70 in all the species tested. In fields, baiting with the same loose bait (0.025%) was performed in the live burrows and 88.89% control recorded on the 4th day after treatment. In another trial, three large hay stacks situated near wheat, barley and mustard crops were also treated with difethialone (0.025%) and 90.47,94.44 and 80.00% kills were recorded respectively. The results of the present investigation prove high potency and acceptability of difethialone against both domestic as well as field rodents.