To screen gram-negative bacteria in the midguts of lab-reared Anopheles sinensis at 3 development stages (larva, unfed adult and engorged adult).Twenty engorged adult An. sinensis mosquitoes were dissected, and bacteria were isolated from their midguts by plate cultivation. The bacteria were identified by using 16S rDNA V3 sequence and dyed with Gram-reaction solutions. Using denaturing gradient gel electrophoresis (DGGE), 16S rDNA clone libraries from common bacteria in the midguts of lab-reared An. sinensis at 3 development stages (20 engorged adults, 10 larva and 50 unfed adults) were constructed. Target Gram-negative bacteria were screened by phylogenetic analysis of all 16S rDNA V3 sequences.In engorged adult female An. sinensis, 16S rRNA sequencing placed the 28 screened bacterial colonies with their closest matches into 5 major groups. Except Gram-positive Brevibacillus sp., the other four, Aeromonas sp., Comamonas sp., Elizabethkingia sp., and A4 were Gram-negative. DGGE analysis showed that 4 distinct bands existed in 3 development stages of An. sinensis. 16S rDNA V3 segments revealed 5 species of bacteria which were Aeromonas sp., A4, Pseudomonas sp., Leucobacter sp., and one unclassified bacterium. Compared with 16S rDNA V3 region of Aeromonas sp. (GQ301543) and A4(FJ8701127), the matching 16S rDNA segments of midgut bacteria of engorged adults showed a nucleotide sequence similarity of 100%.Aeromonas sp. and A4 were the common midgut Gram-negative bacteria of An. sinensis at 3 development stages.