100S is a new thermo-sensitive genic male sterile line of Brassica napus. Two rapeseed inbreeding lines(09157and 09254), three thermo-sensitive genic male sterile lines(Xiangyou 402S, 104S and Z094S) and four recessive genic male sterile lines (402AB, 104AB, 209AB and 127AB) were used for the test crossings with 100S and genetic analysis. The results showed that the fertility of 100S was controlled jointly by two pairs of recessive male sterile genes and two pairs of main effective thermo-sensitive genes, there was no effect of the cytoplasm in 100S. The fertility of 100S and Xiangyou 402S, 104S and Z09S was regulated by different genetic systems. Their male sterile genes were not allelic. The thermo-sensitive genes of 100S couldn’t regulate the male sterile genes of Xiangyou 402S, 104S and Z09S, and the thermo-sensitive genes of Xiangyou 402S, 104S and Z09S had no effects on 100S infertility genes too. The male sterile genes of 100S and the male genes of recessive genic male sterile line 127A were allelic. The thermo-sensitive genes of 100S could control the fertility of 127A. The above conclusions were further validated by the investigation of inheritance in the offspring from the combination ‘100S×127B’.