Objective To investigate the relationship between uridine diphosphate-glucuronosyl transferase 1A1 (UGT1A1), organic anion transporter family member 1B1 (SLCO1B1) and glucose-6-phosphate dehydrogenase (G6PD) gene polymorphisms and neonatal hyperbilirubinemia in southwest China. Methods A total of 190 neonates with neonatal hyperbilirubinemia from September 2016 to December 2019 in Yunnan, Guizhou and Sichuan provinces were selected as the study group, and 200 neonates without jaundice who were hospitalized during the same period were randomly selected as the control group. Genomic DNA was extracted from venous blood and 17 SNPs sites were genotyped by Sequenom-specific SNP detection. Genotype and allele frequency were compared between the study and control groups. Results There were 190 patients (98 boys and 92 girls) in the study group, and the average gestational age was (38.7±1.2) weeks. In the control group, there were 200 neonates (116 boys and 84 girls), and the average gestational age was (38.9±1.3) weeks. The allele frequencies of UGT1A1 rs873478, rs34946978 and rs4148323 were higher in the study group than those in the control group, while the allele frequency of SLCO1B1 rs72559748 was lower in the study group than that in the control group, and the differences were statistically significant (P<0.05). The G6PD rs72554664 variant was detected in two boys with hyperbilirubinemia, and the detection rate was 1.05%. There was significant difference in the distribution of risk alleles between the study group and the control group (P<0.05), and the proportion of risk alleles ≥2 was higher in the study group. Conclusions The UGT1A1 rs873478, rs34946978 and rs4148323 polymorphisms were associated with higher risk of neonatal hyperbilirubinemia in southwest China. The SLCO1B1 polymorphism was not significantly associated with the incidence of neonatal hyperbilirubinemia and lack of G6PD enzyme activity may not be a major risk factor for neonatal hyperbilirubinemia in this region.