BACKGROUND Fifteen lactating Holstein dairy cows were assigned to three diets in a 3 × 3 Latin square design to evaluate the effects of dietary forage sources on rumen microbiota, rumen fermentation and biogenic amines. Diets were isonitrogenous and isocaloric, with a forage/concentrate ratio of 45:55 (dry matter basis) but different main forage sources, namely cornstalk (CS), Leymus chinensis (LC) or alfalfa hay (AH). RESULTS Pyrosequencing of the V3-V6 hypervariable coding region of 16S rRNA revealed that the rumen microbiota was significantly affected by forage sources. AH feeding increased the proportion of genera Prevotella and Selenomonas compared with the CS diet, while CS feeding increased the proportion of genera Anaerotruncus, Papillibacter, Thermoactimoyces, Bacillus and Streptomyces compared with the LC or AH diet. AH and LC feeding both increased the propionate concentration compared with the CS diet. AH feeding decreased the concentrations of tyramine, putrescine and histamine compared with the LC diet. CONCLUSIONS These results indicate that a high proportion of alfalfa hay in the ration is beneficial for milk yield and a healthy and balanced rumen microbiota in lactating cattle. This can be attributed to the higher degradation of rumen organic matter and the more balanced carbohydrates and proteins for optimal rumen microbial growth.