This study investigated the effects of three roughage physically effective neutral detergent fiber (peNDF_8.00) levels (11.52%, 13.16%, and 15.43% on an as-fed roughage basis) on growth performance, rumen fermentation, serum biochemical parameters, rumen microbiota, and rumen metabolomic profiles in yaks. Thirty healthy yaks were allocated to three treatment groups (A, B, and C). The roughage peNDF_8.00 treatments did not significantly affect short-term growth performance. Ruminal valerate concentration was significantly higher in group C than in group A (p < 0.05), while the molar proportions of isobutyrate and isovalerate differed significantly among treatments (p < 0.001). Serum aspartate aminotransferase (AST) activity and blood urea nitrogen (BUN) concentration also differed significantly among treatments (p < 0.05). Rumen microbiota analysis revealed treatment-associated differences in Chao1 richness and variation in microbial community structure, while LEfSe identified Bacteroidota and Xylanibacter as exploratory candidate biomarkers of group C. PICRUSt2 analysis suggested treatment-associated variation in predicted microbial functional potential. Untargeted metabolomics identified multiple candidate treatment-associated metabolic features; however, no KEGG pathway remained significant after Benjamini–Hochberg false discovery rate (FDR) correction. Overall, higher roughage peNDF_8.00 was associated with variation in rumen fermentation, microbial community structure, and metabolomic profiles, without a detectable effect on short-term growth performance.