Rapeseed (Brassica napus) is one of the most important oil crops worldwide;however, drought seriously curtails its growth and productivity. Identifying drought-tolerant germplasm is an efficient strategy for addressing water shortages. Here, we phenotyped a panel of 264 B. napus accessions at full-bloom stage using water loss ratio (WLR) as drought-tolerant index. It identified 8 low-WLR and 6 high-WLR accessions, regarded as drought-tolerant and drought-sensitive, respectively. Comparing with drought-sensitive accessions at the seedling stage, drought-tolerant accessions had shown better performance in maintaining fresh and dry weights, and performed the higher expression of drought-induced marker genes under drought stress. Subsequently, a total of 139 SNPs (single nucleotide polymorphisms) were identified associated with the WLR using a genome-wide association study (GWAS) among 264 B. napus accessions, with the largest number SNPs at chromosome A10, and 13 SNPs significantly were associated with the WLR (–log10 (p-value)>6). Furthermore, 4 putative candidate genes (BnaC09.RPS6, BnaC09.MATE, BnaA10.PPD5 and BnaC09.Histone) were screened involving in drought tolerance in B. napus. Together, our results highlight the WLR's importance in drought tolerance and establish the foun-dation for improving WLR-associated drought tolerance in rapeseed.