Among leukemia patients, a significant number of deaths are due to Candida septicemia, many of which are associated with previous oral infections. Oral candidiasis detection methods vary, and the relationship between oral candidiasis and Candida colonization (CC) is not well defined. The main objectives of this study were to compare the incidence of CC in a healthy and leukemic population, and also to evaluate the efficacy of three simple and inexpensive methods of detecting oral CC in predicting the occurrence of oral candidiasis. A secondary objective was to portray speciation in the examined populations. Forty-two pediatric leukemia patients and 42 healthy, age-, race-, and gender-matched control patients participated in this study. The three methods of detection were cytological examination of the oral mucosa, and direct culture methods from mucosal smears using Sabouraud's dextrose agar (Becton Dickinson Microbiology Systems, Cockeysville, MD) and Oricult-N (Orion Diagnostica, Espoo, Finland). This study demonstrated an increased prevalence of CC in pediatric leukemia patients with the direct culture method detecting CC in a significantly greater proportion of the population (Oricult-N,P = 0.034; Sabouraud's dextrose agar, P = 0.0036). Candida albicans was the predominant species. Further study is needed to determine the clinical significance of oral CC and its relationship to oral candidiasis and systemic infection in pediatric leukemia patients.