In this work 20 clinical and 3 environmental yeast isolates were characterized by classical morphological and physiological methods, as well as molecular methods based on PCR of the ITS1-5.8S rDNA-ITS2 region. The characteristic morphology and biochemical profiles observed in these samples correspond to those described for the Pichia genera, more specifically to P. anomala. The profiles of susceptibility to five antifungal drugs were determined by two broth dilution methods. The results obtained by both methods were comparable and showed that clinical isolates presented more resistance to azoles, amphotericin B, and 5-fluorocytosine, than environmental ones did. By amplification and sequencing of internal transcribed spacers (ITS1 and ITS2) and the ribosomal 5.8S DNA, the yeast samples were divided into four groups, where the strains within each group had the same sequence. Of the analyzed yeast isolates, 78% were identified as Pichia anomala. Using RAPD analysis with seven different Operon primers, polymorphism was observed within the four groups. Our study highlights the growing importance of P. anomala in fungemic episodes in premature neonates. Furthermore, the methodologies used provide a powerful tool to identify and determine differences in similar strains of this yeast