The aim of this study was to investigate whether surfactant-associated protein B (SP-B) mRNA deficiency is involved in the pathogenesis of neonatal respiratory distress syndrome (RDS). A total of 60 unrelated neonates who died of RDS were recruited as the RDS group and subgrouped into a ≤32-, a 32-36+6 and a ≥37-week group (n=20 per group) on the basis of gestational age. In addition, 60 neonates who succumbed to other diseases were enrolled as controls. The lung tissues were collected within 30 min after death. In situ hybridization was conducted to detect SP-B mRNA expression in the lung. The frequency of SP-B mRNA deficiency was also calculated. Among the RDS groups, the SP-B mRNA levels were significantly higher compared to those in the control group (t=7.812, P<0.001), but were comparable among RDS patients with different gestational ages (F=2.348, P>0.105). Among the control groups, the SP-B mRNA levels increased with the increase in gestational age (F=50.124, P<0.001). In the ≤32-week group, the number of cells positive for SP-B mRNA in RDS patients was markedly reduced as compared to that of the controls (t=3.185, P<0.01). In the 32-36+6-week group, the number of cells positive for SP-B mRNA in RDS patients was significantly smaller compared to that of the controls (t=9.342, P<0.001). In the ≥37-week group, the number of cells positive for SP-B mRNA in RDS patients was markedly smaller compared to that in the controls (t=4.238, P<0.001). Among RDS neonates, SP-B mRNA deficiency was noted in 35 patients with a frequency of 58.3%. In the control group, SP-B mRNA deficiency was noted in 8 patients with a frequency of 13.3%, which was markedly lower compared to that in the RDS group (χ 2=26.421, P<0.001). The results of the present study therefore suggest that SP-B mRNA deficiency is involved in the pathogenesis of RDS.