Prairie junegrass (Koeleria macrantha) is a native cool-season C3 grass that has shown potential as a low-input turfgrass. An increased understanding of the physiological and molecular responses of prairie junegrass to water-deficit conditions is important for developing cultivars with enhanced drought tolerance. The objective of this study was to characterize the antioxidative responses and candidate gene expression in prairie junegrass subjected to drought stress. Two drought-tolerant (TOL-1 and TOL-2) and two drought-susceptible (SUS-1 and SUS-2) genotypes of prairie junegrass were subjected to 7 days of drought stress. Leaf relative water content (RWC) of SUS-1 and SUS-2 was 72.1% and 73.8% and RWC of TOL-1 and TOL-2 was 90.1% and 85.4% in drought-stressed plants, respectively. Drought stress did not affect chlorophyll fluorescence, lipid peroxidation, and antioxidative enzyme activities of superoxide dismutase (SOD), catalase (CAT), peroxidase, ascorbate peroxidase (APX), or glutathione reductase for tolerant or susceptible genotypes. The TOL-2 and SUS-2 genotypes were further examined for candidate gene expression. Drought stress did not alter expression levels of CAT and chloroplastic copper/zinc SOD (Cu/ZnSOD), but increased levels of APX in either genotype, compared with their relative controls. Expression of P5CS encoding Δ1-pyrroline-5-carboxylate synthetase and P5CR encoding Δ1-pyrroline-5-carboxylate reductase for proline biosynthesis were up-regulated under drought stress for both genotypes; however, expression of P5CR was more strongly induced under drought stress for TOL-2, compared with its control. The expression of 1-FFT encoding fructan:fructan 1-fructosyltransferase, which is involved in fructan biosynthesis, was strongly induced under drought stress for TOL-2 but not detected under either control or drought stress conditions for SUS-2. These results indicate that the genes involved in proline and fructan biosynthesis may play an important role in drought tolerance in prairie junegrass.