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Background: Vaginal candidiasis is common in during pregnancy. It may lead to complications like abortions, premature birth, low birth weight, chorioamnionitis and fungal systemic neonatal infection. The aim of present study was identification of Candida species by mycological and molecular methods in pregnant women with vaginal candidiasis. Methods: This cross-sectional study was performed on 80 pregnant women with or without clinical symptoms of vulvovaginal candidiasis referred to Shahid Noorani Talesh Hospital, Gilan University of Medical Sciences, Iran, from April to December 2015 (8 months). All specimens were examined by direct microscopy and culture on CHROMagar Candida medium for isolation and differentiation of major clinical-significant Candida species (spp.). Cultured media were incubated at 35 °C for 48 hours and evaluated based on color and number of grown colonies. If no growth was observed, the media were incubated for several additional days. Subcultures were done on Sabouraud dextrose agar (Merck, Germany) and Corn meal agar with Tween 80 media (Micromedia, Hungary) for further study. Identification of Candida spp. carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Results: In this study, vulvovaginal candidiasis was observed in 20 (25%) patients. Twenty-two isolates were obtained from culture of specimens on CHROMagar Candida medium (Paris, France). The most common isolated species was Candida albicans 16 (72.8%) and followed by Candida glabrata 5 (22.7%), Candida tropicalis 3 (13.6%) and Candida krusei 1 (4.5%) cases. Two patients had mixed infection with 2 different Candida species (C. albicans and C. glabrata) While using PCR-RFLP method, the Candida species were identified as 13 (59.1%) Candida albicans, 5 (22.7%) Candida glabra, 3 (13.6%) Candida tropicalis and 1 (4.5%) Candida krusei cases, respectively. In direct examination were seen yeast budding cells and pseudohyphae in 8 culture positive specimens. In the present study, results of conventional mycological method in differentiation of Candida spp. were consistent with molecular results in 80% of cases. There was also significant correlation between vulvovaginal candidiasis with clinical symptoms (P