Sporotrichosis is a subcutaneous mycosis worldwide distributed reaching hyperendemic proportions in Brazil. Many isolates from patients with sporotrichosis are preserved in culture collections by different methods around the world. The preservation methods are used to maintain the viability and the morphophysiological and genetic characteristics of isolates for long periods. In this study, we evaluated 34 isolates, previously, identified as S. schenckii by a classical identification method, initially preserved by periodical subcultures and then under mineral oil at culture collection of Oswaldo Cruz Institute/Fiocruz, to re-identify them by polyphasic identification. Our results showed that seven isolates remained viable for 34 to 64 years under oil, one isolate lost the ability to sporulate which was reverted by using a medium culture supplemented with rosebush branches and all of them were identified as Sporothrix schenckii sensu stricto by morphological, physiological, partial β-tubulin gene sequencing and phylogenetic analysis.