[Objective] A molecular-based approach, terminal restriction fragment length polymorphism (T-RFLP), for community analysis of ruminal methanogens was developed and used to assess the effects of transfer frequency on the community of methanogens co-cultured with anaerobic fungi. [Methods] The specific primers for mcrA genes were used to amplify the mcrA genes of methanogens and the amplicons were then digested with restriction enzymes. The size of each of the individual resulting terminal fragments were detected using DNA sequencer. [Results] With the analysis by Msp I, the dominant methanogens co-cultured with anaerobic fungi were those with terminal fragment of 470 bp and those of 130 bp and 200 bp were also dominant in the 15th transfers of 3-day co-cultures. Comparative study with Taq I showed that the dominant methanogens both in rumen digesta and 3-day co-cultures were those with terminal fragment of 70, 100, 200, 270, 300, 330 and 470 bp. The methanogens of 70, 100, 270 and 470 bp changed dramatically during in vitro transfers. Subsequently, the effects of transfer frequency on community of methanogens co-cultured with anaerobic fungi were assessed by Taq I and results showed that the community of methanogens in 3-day co-cultures was similar with rumen digesta, while they are significantly different from those in 5- and 7-day co-cultures, which was resulted from the shift of methanogens with terminal length of 100, 70 and 270 bp. [Conclusion] The molecular-based approach, T-RFLP, was suitable for analysis of ruminal methanogens. The community of methanogens co-cultured with anaerobic fungi was significantly affected by transfer frequency of the co-cultures, and that in 3-day co-cultures was similar to that in the rumen digesta.