In this work the actin gene was used to establish phylogenetic relationships of wider and more diffuse species of the genus Saccharomyces in food ecology by temporal temperature gradient electrophoresis (TTGE) and amplified restriction fragment length polymorphism ( RFLP) analysis. Results for DNA RFLP analysis varied considerably, and some enzymes showed a high intra- and interspecific power; however, comparison of experimental results with those provided by the National Center for Biotechnology Information database disclosed a number of interesting variations. Only some experimental results matched the theoretical ones. A theoretical study of melting temperatures using available information from partial sequences of the actin gene was done. Several Saccharomyces species and strains could be distinguished using different TTGE melting points. Some degree of discrimination was achieved under different conditions, in that the Saccharomyces strains tested were separated into groups like the results obtained by PCR-RFLP.