The study of neonatal gender differences in pain expression is important since neonatal pain behavior occurs prior to any learned reaction pattern. The objective of this study was to verify the presence of gender differences in pain expression in preterm and term newborn infants. Sixty-five consecutive neonates (37 female and 28 male infants) with gestational age between 28 and 42 weeks and with 25-120 h of life were studied. Healthy term neonates required a capillary puncture for PKU screening and clinically stable premature infants needed a capillary puncture for glucose dosage. The Neonatal Facial Coding System (NFCS) and the Neonatal Infant Pain Scale (NIPS) were evaluated at bedside prior to the puncture, when patients were at rest, during foot heating; during capillary puncture; and at 1, 3, and 5 min after heel lancing. Results were analyzed by repeated-measures ANOVA followed by the Multiple Comparison Method of Bonferroni. A significant difference among the mean NFCS scores during the six study periods was noted for the whole group of neonates (P<0.000001). Also, a significant interaction between the NFCS score profile in female and male neonates at the different study periods was observed (P=0.025). Regarding NIPS, ANOVA showed only a significant difference among the mean NIPS scores during the six study periods for the whole group of neonates (P<0.000001). No significant interactions between gestational age and time, nor between gestational age and gender were noted, for both NFCS and NIPS. In conclusion, recently born female neonates of all gestational ages expressed more facial features of pain than male infants, during the capillary puncture and 1 min afterwards. Maybe differences in pain processing and/or pain expression among genders may explain this finding.