Three major clusters of mitochondrial tRNA genes (tRNA(Ile) -tRNA(Gln) -tRNA(Met), tRNA(Trp)- tRNA(Ala) -tRNA(Asn)- tRNA(CYs) -tRNA(Tyr) and tRNA(His) tRNA(Ser)(AGY) -tRNA(Leu)(CUN) from 13 species of Predatory birds were amplified and sequenced. The length of these tRNA clusters was similar among species (212 approximately 214 bp, 353 approximately 362 bp, 205 approximately 208 bp, respectively), and 47% of the sequences were variable, 67% of which were involved in the loop regions. The stem regions were relatively conserved, and the variable base pairs were under the restriction of compensatory changes or G-U wobble pairing which could be regarded as mechanisms for maintaining a stable secondary structure. Maximum-parsimony (MP) and Neighbor joining (NJ) phylogenetic trees were constructed using all the tRNA gene sequences or stein-forming nucleotides with Caprimulgus indicus as outgroup. We found that the bootstrap values for branches of trees using the tRNA sequences were commonly higher than the others, therefore the phylogenetic relationship of Predatory birds reflected by these data may be closer to the truth. Phylogenetic analyses indicated that Accipitridae was closer to Strigidae instead of Falconidae, and the classification of Tytonidae was different from the conclusion from the previously morphological and DNA-DNA hybridization studies. By comparing the secondary structure among taxa we found that the characters of nucleotide insertions and deletions in some tRNA genes have synapomorphies, suggesting that these characters may be useful for resolving the phylogenetic relationship of different families in Predatory birds with higher phylogenetic performance.