Quinoa (Chenopodium quinoa Willd.) has rich nutritional value and strong adversity resistance. In this study, two germplasm materials, "M059" and "M024", showing with fast and slow radicle growth, were treated by PEG-6000 hypertonic solution method to simulate drought stress, and comprison groups of C1vsD1, C2vsD2, C1vsC2, and D1vsD2 were adopted for RNA-seq analysis. The radicle length of M059 and M024 decreased by 68.65% and 71.43% respectively under 15% PEG treatment for 24h compared with water treatment for 24h. The contents of total soluble sugar, sucrose, glucose and fructose in M059 were 18.58%, 97.84%, 70.54% and 32.77% higher than those in M024 under water treatment for 24 h. The content of sucrose in M024 was 23.01% higher than that of M059 under 15% PEG treatment for 24h. The contents of total soluble sugar and glucose in M059 were 7.26% and 25.00% higher than those in M024, respectively, under 15% PEG treatment for 24h. Transcriptome sequencing results showed that 7042 differentially expressed genes (DEGs) were obtained under drought stress treatment. Venn analysis showed that there were a total of 211 commonable DEGs and 132, 1270, 578 and 914 unique DEGs in the four comparison groups. GO and KEGG enrichment analyses showed that five GO Terms and three pathways were closely related to the molecular response of glucose metabolism in quinoa seeds under drought stress. The qRT-PCR results of the 10 key candidate genes (LOC110702784_AGAL2、LOC110719866_INV1、LOC110717843_TPPJ、 LOC29490_CELB、LOC110719843_bg1x、LOC110689796_SUS1、LOC110690728_MAN6、LOC110729879_HK2、LOC110712726_EGLC、 LOC110734349_FK7) were expressed consistent with that of the transcriptome results. The results of this study will provide reference for further analysis of the molecular regulatory mechanism under drought stress in quinoa.