The salivary gland polytene chromosomes of Simulium qianense was observed with wild larvae collected from Guiyang, Guizhou Province. Using an upgraded method of preparation, the polytene chromosomes of the larval salivary glands of S. qianense were observed and analysed. The results showed three pairs of chromosomes (2n = 6) arranged from the longest to the shortest. Chromosome I was metacentric while chromosomes II and III were submetacentric. The centromeric regions distinctly expanded and were easily recognized. The nucleolar organizer situated near the centromeric band of the short arm of chromosome II. The Balbiani ring and double bubble all located near the tip of the short arm of chromosome II. There were inversions in some individuals, with an inversion frequency of 33% (32/97). The study revealed constant localization and morphology of the principal landmarks (namey, centromere, nucleolar organizer, Balbiani ring, double bulge, band and puff) which can be considered as distinguishable features of S. qianense.