Library of ruminal protozoal 18S rRNA of Yunnan Yellow Cattle has been constructed in the present study. Phylogenic analysis of sequences was meanwhile employed to reveal the diversity of protozoa in the rumen of Yunnan Yellow Cattle. One Yellow Cattle was fed malt meal (YCRPB) and the other was fed wheat straw (YCRPS). A protozoa-specific primer (P-SSU-342f) and a eukarya-specific primer (Medlin B) were used to amplify a 1,360-bp fragment of DNA encoding protozoal small subunit (SSU) ribosomal RNA from rumen fluid. The results showed as follows: A total of 121 clones were obtained and fell into four genera identified as Entodinium (66.9%), Dasytricha (5.8%), Isotricha (9.1%), and Diplodinium (18.2%). Within the genus Entidinium, 48 of the YCRPB sequences and 33 of the YCRPS sequences clustered with the Entodinium caudatum. 7 of the YCRPB sequences were phylogenetically placed within the genus Dasytricha. 11 of the YCRPB sequences were related with high confidence to Isotricha intestinalis. 22 of the YCRPS sequences were phylogenetically placed within the genus Diplodinium. The predominant protozoal genus identified in the rumen fluid belonged to the Entodinium group, and the divergences between two cattle may due to diet and individual differences.