Thousand-seed weight of Brassica napus is one of most important yield traits. Construction of the genetic linkage map will provide the basis for research on the genetic base of quantitative trait in Brassica napus. By microspore culture technology, we screened the pure lines DH-G-42 with large seed and DH-7-9 with small seed, their thousand-seed weights were 6.24 g and 2.42 g, respectively, and the ratio of 1000-seed weight for two lines is 2.58. A genetic linkage map with F2 population including 190 individuals derived from a cross of DH-G-42  DH-7-9 were established by SSR and SRAP markers. The map consisted of twenty linkage groups anchored 128 SSR and 100 SRAP markers and covered 1546.6 cM with an average marker interval of 6.78 cM. QTLs located in A9 and C1 linkage group were identified to be significant for the 1000-seed weight trait. The qSW-A9-1 and qSW-A9-2 explained 10.98% and 27.45% of the phenotypic variation, which could be considered as main effect QTL for the 1000-seed weight trait. Our study will be beneficial to the fine QTL mapping, the marker-assisted breeding and the novel genes cloning in Brassica napus.