Over the last decades, there have been important changes in the epidemiology of Candida infections. In recent years, Candida species have emerged as important causes of invasive infections mainly among immunocompromised patients. This study analyzed Candida spp. isolates and compared the frequency and biofilmproduction of different species among the different sources of isolation blood, urine, vulvovaginal secretions and peritoneal dialysis fluid. Biofilmproduction was quantified in 327 Candida isolates obtained from patients attended at a Brazilian tertiary public hospital (Botucatu, Sao Paulo). C. albicans ALS3 gene polymorphism was also evaluated by determining the number of repeated motifs in the central domain. Of the 198 total biofilm-positive isolates, 72 and 126 were considered as low and high biofilm producers, respectively. Biofilmproduction by C. albicans was significantly lower than that by non-albicans isolates and was most frequently observed in C. tropicalis. Biofilmproduction was more frequent among bloodstream isolates than other clinical sources,in urine, the isolates displayed a peculiar distribution by presenting two distinct peaks, one containing biofilm-negative isolates and the other containing isolates with intense biofilmproduction. The numbers of tandem-repeat copies per allele were not associated with biofilmproduction, suggesting the evolvement of other genetic determinants.