Winter turnip rape (Brassica rapa L.) is an important overwintering oil crop that is widely planted in northwestern China. It considered to be a good genetic resource for cold-tolerant research because its roots can survive harsh winter conditions. Here, we performed comparative transcriptomics analysis of the roots of two winter turnip rape varieties, Longyou7 (L7, strong cold tolerance) and Tianyou2 (T2, low cold tolerance), under normal condition (CK) and cold stress (CT) condition. A total of 8,366 differentially expressed genes (DEGs) were detected between the two L7 root groups (L7CK_VS_L7CT), and 8,106 DEGs were detected for T2CK_VS_T2CT. Among the DEGs, two [omega]-3 fatty acid desaturase (FAD3), two delta-9 acyl-lipid desaturase 2 (ADS2), one diacylglycerol kinase (DGK), and one 3-ketoacyl-CoA synthase 2 (KCS2) were differentially expressed in the two varieties and identified to be related to fatty acid synthesis. Four glutamine synthetase cytosolic isozymes (GLN), serine acetyltransferase 1 (SAT1), and serine acetyltransferase 3 (SAT3) were down-regulated under cold stress, while S-adenosylmethionine decarboxylase proenzyme 1 (AMD1) had an up-regulation tendency in response to cold stress in the two samples. Moreover, the delta-1-pyrroline-5-carboxylate synthase (P5CS), [delta]-ornithine aminotransferase ([delta]-OAT), alanine-glyoxylate transaminase (AGXT), branched-chain-amino-acid transaminase (ilvE), alpha-aminoadipic semialdehyde synthase (AASS), Tyrosine aminotransferase (TAT) and arginine decarboxylase related to amino acid metabolism were identified in two cultivars variously expressed under cold stress. The above DEGs related to amino acid metabolism were suspected to the reason for amino acids content change. The RNA-seq data were validated by real-time quantitative RT-PCR of 19 randomly selected genes. The findings of our study provide the gene expression profile between two varieties of winter turnip rape, which lay the foundation for a deeper understanding of the highly complex regulatory mechanisms in plants during cold treatment.