Background: We examined selected polymorphisms in 3 pulmonary surfactant-associated proteins (SP) for their influence on serum SP levels and risk of respiratory distress syndrome (RDS) in preterm neonates. Material/Methods: Premature infants from a Han population were enrolled, including 100 premature infants with RDS (case group) and 120 premature infants without RDS (control group). SNP genotyping for SP-A (+186A/G and +655C/T), SP-B (-18A/C and 1580C/T), and SP-D (Met11ThrT/C and Ala160ThrG/A) used polymerase chain reaction-restriction fragment length polymorphism. Haplotypes were calculated with Shesis software and serum SP-A/B/D levels were quantified by ELISA. Results: Case and control groups exhibited significant differences in genotype and allele frequencies of SP-A (+186A/G, +655C/T) and SP-B (1580C/T). However, no statistically significant differences were observed in the allele and genotype frequencies of SP-B -18A/C, SP-D Met11ThrT/C, and SP-D Ala160ThrG/A. Importantly, serum SP-A and SP-B levels were reduced in RDS patients carrying SP-A (+186A/G, +655C/T) and SP-B (1580C/T) polymorphisms. AA genotype of +186A/G, SP-A level, and CC genotype of 1580C/T were independently correlated with increased RDS risk. Conclusions: SP-A (+186A/G) and SP-B (1580C/T) polymorphisms are strongly associated with the risk of RDS in preterm infants. Notably, reduced serum SP-A levels were correlated with a high risk of RDS and may serve as novel biomarkers for RDS detection and monitoring.