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Celis-Ramírez A.M., Guevara-Suarez M., Galvis-Marín J.C., Rodríguez-Bocanegra M.X., Castañeda-Salazar R., Linares-Linares M.Y., Triana-Sierra S.H. & Pulido-Villamarín A.P. 2019. Genotyping of Malassezia pachydermatis disclosed genetic variation in isolates from dogs in Colombia. Pesquisa Veterinária Brasileira 39(11):915-922. Unidad de Investigaciones Agropecuarias, Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Carrera 7 43-82, Ed. 52, Of. 608, Bogotá, Colombia. Departamento de Ciencias Biológicas, Universidad de Los Andes, Carrera 1 18A-10, Bloque J, Of. J-214, Bogotá, Colombia. E-mails: adriana.pulido@javeriana.edu.co, acelis@uniandes.edu.co Malassezia pachydermatis is a lipophilic and lipid-dependent yeast mostly isolated from animals’ skin; hence, it is regarded as a zoophilic species causing otitis externa in dogs. Aspects associated with its epidemiology and pathogenicity is a matter of interest. This study aimed to conduct a molecular characterization of 43 isolates of M. pachydermatis obtained from dogs with otitis externa. For this purpose, the 5.8S internal transcribed spacer 2 (ITS2) and D1/D2 26S rRNA regions were amplified, sequenced and analyzed using restriction fragment length polymorphism (RFLP) with AluI, CfoI, and BstF5I endonucleases. Phylogenetic analyses revealed that these isolates grouped with the sequence types I, IV and V, previously proposed for M. pachydermatis. Interestingly, we found a new polymorphic RFLP pattern using BstF5I, these isolates were associated with the sequence types IV and V, nevertheless an association between polymorphic RFLP patterns, and fosfolipase activity or canine population data was not observed. These findings underline the genetic diversity of M. pachydermatis and provide new insights about the epidemiology of this species in the analyzed population.