Eighteen pathogenic native strains of Bacillus sphaericus that were pathogens to mosquito larvae were isolated from different Colombia regions. The objective of this study was to evaluate at physiological and molecular level pathogenic strains and to compare them with the reference 2362 one. Cellular growth, sporulation percentage, pathogenic activity against third instar larvae of Culex quinquejasciaius, the presence of toxigenic proteins, the size of native plasmids and the genetic polymorphism among pathogenic and non pathogenic isolations was evaluated. The evaluated strains of Bs presented a latency stage ranging from 2 to 3 h and one logarithmic phase of 7 h; the sporulation in BHI was lower than 1% to 40 h of incubation, in NYSM medium was obtained ten times more production of biomass and spores, 26% of the population showed sporulation percentage higher than 90%; the isolations were classified in three groups of pathogenicity with [Formula] and [Formula]. Punctual mutations on the genes which encoding for native toxins were detected and was found a exclusive protein of 30 kDa in pathogenic native strains. The isolations of Bs presented a plasmid of 118Kb that was not related to the toxicity; pathogenic strains are a homogenous group with a similarity between 90 to 100%, whereas non-pathogenic ones are genetically heterogeneous group and conform a cluster aside. The native strains have a great potential in biological control of mosquito larvae that transmitting diseases such as dengue, malaria, encephalitis and filariasis among others.