Background and Aims: This study was done to detect the prevalence, risk factors for vaginal candidiasis in Chennai and to evaluate different methods for speciation of Candida isolates from vaginal candidiasis patients. This study was also aimed at detecting resistance patterns of Candida spp to common antifungals and at detecting mutant FUR1 genes in 5-Flucytosine (5 FC) resistant isolates. Material and Methods: Two hundred clinically suspected vaginal candidiasis patients were screened for candidiasis and isolated Candida were speciated by standard morphological and biochemical tests (sugar fermentation and assimilation) and by using CHROM agar-Candida medium. Antifungal susceptibility was performed by disk diffusion method (CLSI M44-A) using fluconazole, itraconazole and 5FC disks. Five FC resistant isolates were subjected to PCR for detection of mutant FUR1 genes. Results: A total of 72 (36%) Candida spp. were obtained. Vaginal candidiasis was more prevalent in 31-40 years age group and among those with poor genital hygiene and who wore tight fitting synthetic/nylon underclothes. C. albicans (35), C. tropicalis (8), C. glabrata (21), C. krusei (4) were identified by both carbohydrate assimilation test and by using CHROM agar-Candida medium. C. kefyr (2) and C. parapsilosis (2) could not be identified using CHROM agar-Candida. Resistance to fluconazole, itraconazole and 5-flucytosine was seen in 19.44%, 23.61% and 41.66% of the isolates respectively. Mutant FUR1 gene was detected in all the Candida spp that were resistant to 5FC. Conclusion: C. albicans was the commonest species which caused vaginal candidiasis in Chennai. Though CHROM agarcandida medium is a useful differential isolation medium capable of early presumptive identification of Candida species, it could not identify C. kefyr and C. parapsilosis. Azole resistance was low in C. albicans but it was high in non-albicans Candida spp. Prevalence of primary resistance to 5-flucytosine was high in the strains studied and in all of them, it was mediated by mutant FUR1 gene.