Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common inborn enzymatic defect in the world. Determining the prevalence of G6PD deficiency is an essential step towards evaluating its impact on the health of a population. Therefore, this study aimed to determine the prevalence and factors associated with G6PD deficiency among newborns in Sana'a city, Yemen. Subjects and methods: This cross-sectional study included 397 newborns from eight public and private hospitals in Sana'a city in the period from January to March 2020. Data about gender and the factors possibly associated with G6PD deficiency were collected using a pre-designed questionnaire. G6PD deficiency was qualitatively detected in fresh blood using rapid diagnostic tests (RDTs). Data were analyzed using SPSS software, and differences were considered statistically significant at p-value <0.05. Results: Of 397 screened newborns (217 males and 180 females), 19 newborns (14 male and 5 female newborns) were G6PD-deficient, with an overall prevalence of 4.8% and a male: female ratio of 2.8:1. G6PD deficiency was significantly associated with consanguinity. However, it was not significantly associated with the gender, mode of delivery, gestational age or birth weight of the newborns. Conclusions: G6PD deficiency is prevalent among as low as approximately 5.0% of newborns in the hospitals of Sana'a city. It is almost comparable to those reported from other countries in the region. It is more prevalent among males than females, with no statistically significant difference. G6PD deficiency was not associated with the gender, mode of delivery, gestational age and birth weight of the newborns.