Yeast isolates from orange fruit and juice in a spontaneous fermentation were identified and classified by two molecular techniques. The first was analysis of the restriction pattern generated from the polymerase chain reaction (PCR)‐amplified 5.8S rRNA gene and the two internal transcribed spacers (ITS) using specific primers. The second technique was sequence analysis of the ITS regions using the same two primers. Nine different restriction profiles were obtained from the size of the PCR products and the restriction analyses with three endonucleases (CfoI, HaeIII and HinfI). These groups were identified as Candida tropicalis, Clavispora lusitaniae, Hanseniaspora uvarum, Pichia anomala, Pichia fermentans, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Saccharomyces unisporus, and Trichosporon asahii. Checking against identification according to morphological, physiological and biochemical traits corroborated this molecular identification. A total concordance was found in the identification with PCR‐restriction fragment length polymorphism of the ITS region after analysing certified yeast strains from two different culture collections. Consequently, a rapid and reliable identification of the yeast populations was achieved by using molecular techniques.