Understanding the natural diets of grazing herbivores can help fulfill their nutritional requirements and develop management strategies. Emerging metabarcoding techniques can provide more accurate estimates for dietary composition of grazing animals. Thirty-nine Tan sheep with weights of 25.10 ± 1.88 kg were randomized into three groups: the grazing group, the time-limited grazing group, and the stall-fed group. Effects of grazing on meat fatty acid composition in lambs were compared to concentrate-based systems. Simultaneously, we investigated sheep diets using DNA metabarcoding of feces to assess the prevalence of medicinal herbage plants consumed by grazing sheep. Metabarcoding data determined that Lespedeza sp., Artemisia sp., Chenopodium sp., Corispermus sp., and Phellodendron amurense were predominant with different proportions (P < 0.05). Our results demonstrated that grazing systems could transform the muscle fatty acid composition and promote n-3 PUFAs, including C18:3n3 (ALA), C20:5n3 (EPA), and C22:6n3 (DHA) deposition. To establish the association of PUFAs with the herbage taxa, we conducted multivariate and correlation analyses. Some highlighted herbage species (e.g., Bassia scoparia, Euphorbia humifusa, and Arnebia euchroma) were significantly correlated with omega-3 PUFAs. The dominant group Lespedeza sp. showed a positive correlation with C18:2n6. Overall, these results demonstrated the utility of metabarcoding diet analysis and how diversification in dietary composition was associated with muscle fatty acid deposition. This research examined the correlation between herbage taxa and omega-3 fatty acids, and the results provide an initial view of the effects of herbage on PUFAs of lambs. The study provides experimental evidence for future feeding research.