Candidemia remains a major cause of morbidity and mortality in the health care environment. The epidemiology of Candida infection is changing, mainly in relation to the number of episodes caused by species C. non-albicans. The overall objective of this study was to evaluate the frequency of yeasts of the genus Candida, in a four-year period, isolated from blood of pediatric patients hospitalized in a public hospital of the city of São Paulo, Brazil. In this period, yeasts from blood of 104 patients were isolated and, the identified species of Candida by phenotypic and genotypic methods were: C. albicans (39/104), C. tropicalis (25/104), C. parapsilosis (23/104), Pichia anomala (6/104), C. guilliermondii (5/104), C. krusei (3/104), C. glabrata (2/104) and C. pararugosa (1/104). During the period of the study, a higher frequency of isolates of C. non-albicans (63.55%) (p = 0.0286) was verified. In this study we verified the increase of the non-albicans species throughout the years (mainly in 2009 and 2010). Thus, considering the peculiarities presented by Candida species, a correct identification of species is recommended to lead to a faster diagnosis and an efficient treatment.