One of the eco-friendly approaches to control mosquitoes is by the use of <i>Lysinibacillus sphaericus</i>. <i>L. sphaericus</i> (previous name <i>Bacillus sphaericus</i>) C3-41 (CP000817) was isolated from Rabbanahalli village of Yadgir district, Karnataka, India and was identified by biochemical and molecular means. Biochemical tests established its identity as <i>L. sphaericus</i>. It is also clear from the phylogenetic analysis that 16S ribosomal RNA nucleotide sequence of <i>L. sphaericus</i> C3-41 (CP000817) showed 99.73 per cent similarity with reference strain. Bioassay of <i>L. sphaericus</i> against second instar larvae of Culex quinquefasciatus recorded significantly higher mortality of 100 per cent at 0.075 % CSM. When assayed against second instar larvae of Anopheles subpictus mortality was 83.33 per cent at 0.075 % CSM.This new strain of L. sphaericus isolated from Northern dry zone of Karnataka, where the temperature ranges from 38°C to 46°C, could be a better biological pesticide to tackle the Culex mosquito and prevent diseases transmitted by it, particularly in Northern Karnataka.