Objective: High prevalence rates of gestational diabetes mellitus (GDM) is a public health concern worldwide. The mechanism that leads to glucose tolerance beyond normal physiological levels to pathogenic conditions remains incompletely understood, and it is speculated that the maternal microbiome may play an important role. The objective of this study is to analyze the gut microbiota composition in each trimester of weight-matched women with and without GDM and examined possible bacterial genera associations. Design: A cohort study. Setting: This study was conducted in an outpatient clinic at the Federal University of São Paulo. Population: 56 women with GDM and 59 without during their first/second or third trimester of gestation. Methods: They were submitted to a standardized questionnaire, dietary recalls, clinical examination, biological samples collection and molecular profile of the fecal microbiota. Results: There was no difference in the alpha diversity and in the overall microbiota structure. A higher abundance of Bacteroides in GDM group was found. A positive correlation between Christensenellaceae and Intestinobacter abundances with 1-hour post-challenge plasma glucose and a negative correlation between Enterococcus and 2-hour plasma glucose level were observed. Bifidobacterium and Peptococcus abundances were increased in the third gestational trimester for both groups. Conclusion: The gut microbiota composition wasn’t dependent on the presence of GDM during the whole gestation. However, some genera abundances showed associations with glucose metabolism. Our findings may motivate deepening the knowledge about physiological and pathophysiological changes in the microbiota throughout pregnancy, which could have further implications for diseases prevention.