Neonatal alloimmune thrombocytopenia (NAIT) is a clinical syndrome that resembles hemolytic disease of the newborn, affecting the platelets only. The thrombocytopenia results from the maternal alloantibodies reacting with specific human platelet antigens (HPAs) on the fetal platelets. Forty-four maternal plasma samples were screened for platelet alloantibodies using qualitative solid phase enzyme-linked immunosorbent assay (ELISA) commercial kit (LIFECODES Pakplus, Hologic Gen-Probe GTI Diagnostics, Waukesha, WI, USA), and both the maternal and the corresponding cord blood samples were genotyped (LIFECODES ThromboType, Hologic Gen-Probe GTI Diagnostics, Waukesha, WI, USA). HPA genotyping results correlated with the genetic frequencies in the Taiwan population. A total of 34 newborns (77.3%) had partial HPA genotyping mismatches with the corresponding mothers. The most common partial mismatches between mothers and neonates in HPA genotypes were 13 (29.5%) in both HPA-3b and HPA-15a, followed by 12 (27.3%) in HPA-15b, and 8 (18.2%) in HPA-3a. The frequencies of homozygotic mother with heterozygotic neonate were 15.9% in both HPA-3a and HPA-15b, 9.1% in HPA-15a, 6.8% in HPA-3b, and 2.3% in both HPA-2a and HPA-6a. In this study, maternal HPA antibodies were found in five samples, whereas HLA class I antibodies were found in seven maternal plasma samples from the antibody screen. The results from this study have demonstrated that HPA mismatch is not the main cause for the production of HPA alloantibodies.