Background: The Beta-3 adrenergic receptor (ADRB3) gene codes for the β3 adrenergic receptor involved in control of energy balance by altering lipolysis and thermogenesis in adipocytes. A missense mutation in codon 64 of this gene caused the replacement Trp with Arg (Trp64Arg/rs4994). A cross-sectional study was performed with the joining of 708 non-obese children and 304 obese children in Hanoi. The objective of our study was to find out the association between rs4994 polymorphism of ADRB3 and obesity in Hanoi preschool-age children, considering their eating behaviors. Results: The frequency of C allele in obese group was 16.4%, higher than that in the control group (11.7%, p = 0.003). After conducting univariate analysis, and multivariate analysis adjusted for eating behaviors, a significant association between rs4994 polymorphism and obesity was observed (p < 0.05). Conclusions: This study indicated that ADRB3-rs4994 polymorphism can be considered as an independent risk factor for obesity in Vietnamese preschool children.