Stevia (Stevia rebuadiana Bertoni) is a perennial shrub grown for the extraction of sweet tasting, nonnutritive compounds called steviol glycosides (SGs) from the leaves, which are up to 300 times sweeter than sugar. The most abundant SGs, stevioside and rebaudioside (Reb) A, have undesirable taste characteristics that have limited their acceptance as a sugar substitute. The SGs Reb D and Reb M offer a similar sweetening intensity to Reb A, but with a superior taste profile. However, these compounds are produced in much lower concentrations than Reb A. The genetic control of SG biosynthesis is only partially understood, hampering efforts to develop new varieties producing high concentrations of desired SGs. Here we report development of a reference transcriptome from a diverse set of stevia tissues. Simple sequence repeat (SSR) markers were mined from the transcriptome, and a genetic linkage map for a pseudo test-cross F1 stevia population was generated with 97 SSRs. This population exhibited transgressive segregation and high broad-sense heritability estimates for many leaf biomass yield and SG content-related traits across two field locations. Two quantitative trait loci (QTL), each explaining 14.0–16.6 % of the observed variation, were identified for Reb D concentration. Additional QTL were identified for Reb A concentration, vigor, and plant height. This study represents the first report of QTL in stevia and will aid future efforts for molecular marker development and gene discovery for economically important traits in stevia.