This study developed and evaluated DNA metabarcoding to identify the presence of pinniped and cetacean prey DNA in fecal samples of East Greenland (EG) and Southern Beaufort Sea (SB) polar bears Ursus maritimus sampled in the spring of 2015-2019. Prey DNA was detected in half (49/92) of all samples, and when detected, ringed seal Pusa hispida was the predominant prey species, identified in 100% (22/22) of EG and 81% (22/27) of SB polar bear samples with prey DNA detected. Bearded seal Erignathus barbatus DNA was found in 19% (5/27) of SB polar bear samples for which prey DNA was detected. Prey DNA detection frequencies and relative abundances were compared to estimates from quantitative fatty acid signature analysis (QFASA) for a subset of SB polar bears. Ringed seal and bearded seal were the main prey identified by both methods, but QFASA also identified 2 cetacean prey species not found by prey DNA. Differences in DNA metabarcoding vs. QFASA results were likely related to the different dietary timescales captured by each approach, i.e. short-term vs. long-term diet, respectively. Prey DNA detection, sex/age class, and subpopulation significantly explained variation in polar bear gut bacterial composition. Polar bear samples with prey DNA detected were associated with higher abundances of the bacterial classes Clostridia and Bacilli and lower abundances of Negativicutes. Fecal DNA metabarcoding is thus useful for identifying recent prey of polar bears, complementing quantitative and likely longer-term QFASA estimates, and may help understand variation in the polar bear gut microbiome.