Because some species of fishes have been becoming more and more difficult to be collected, many phylogenetic researchers have no other choice but adopt formalin-fixed specimens. There are many reports on molecular phylogenetic analyses based on DNA sequences from formalin-fixed specimens, but seldom researches have discussed the reliability about the DNA sequences from formalin-fixed specimens in applying phylogenetic analysis. Here the partial sequences, 1205bp and 1295bp 18S rDNA we reported, were first cloned form muscle of formalin-fixed and live Paralichthys olivaceus by using gene overlap strategy. Compared with live specimen, 80% bases of 18S rDNA sequence are identical, 7% are deleted, and 11% are replaced in formalin-fixed specimen. The DNA sequences from formalin-fixed and live specimens were used simultaneously in phylogenetic analysis based on high homology area. We think it is reliable in high-class molecular phylogenetic analysis.