Rapeseed (Brassica napus) is one of the most important oil crops in the world; however, drought significantly curtails its growth and productivity. Identifying drought-tolerant germplasm is an efficient and low-cost strategy for addressing water shortages. Using water loss ratio (WLR) as an index of drought tolerance, we screened a panel of 265 B. napus lines. We identified eight low-WLR and six high-WLR accessions, which were regarded as drought-tolerant and drought-sensitive, respectively. Further validated these selected accessions at the seedling stage under drought-stress conditions. The drought-tolerant accessions had significantly greater fresh and dry weights under drought stress than the drought sensitive accessions. Using RT-qPCR, we showed that a set of previously reported drought-adaptive marker genes were expressed at higher levels in the drought-tolerant lines than in the drought-sensitive lines. These results indicated that the drought-tolerant genotypes could be identified from natural populations using WLR. Then, we performed a genome-wide association study to identify loci harboring single nucleotide polymorphisms (SNPs). A total of 139 SNPs were significantly associated with the WLR, of which chromosome A10 harbored the largest number. Furthermore, four putative candidate genes were selected by combining the SNP–WLR association results and transcriptional expression data with the changes in drought tolerance. Thus, we have identified two drought-tolerant B. napus cultivars and uncovered genome-wide variation differentiating B. napus lines related to WLR, in addition to providing insights for further research into WLR-related drought mechanisms.