Pre-eclampsia (PE) is a leading cause of maternal and fetal mortality and morbidity. Structural or functional alterations of human leukocyte antigen (HLA)-G present at the maternal-fetal interface may predispose women to PE. We tested the HLA-G gene for association with PE in a case-control study of 83 PE and 240 normotensive Malay women. HLA-G was amplified in a single-tube multiplex-PCR reaction and genotyped for 18 single nucleotide polymorphisms (SNPs) by multiplex-minisequencing. Case-control comparisons were performed, and associations with disease were expressed as odds ratios (ORs). Risk for PE was significantly associated with fetal allele G*0106 only in multigravid pregnancies (P = 0.002, OR = 5.0, 95% CI = 1.8-13.8). Among multigravid pregnancies, the frequency of PE babies heterozygous or homozygous for G*0106 was also significantly higher compared with normal control babies (P = 0.002, OR = 5.4, 95% CI = 1.9-15.4). Multivariate analyses with adjustment for factors associated with PE revealed similar results (P = 0.003, OR = 10.1, 95% CI = 2.2-46.8). Additionally, a significantly higher frequency of fetal-maternal G*0106 genotype mismatch was observed in PE compared with normal multigravid pregnancies (P = 0.001, OR = 9.6, 95% CI = 2.4-38.7). Thus, paternal HLA-G G*0106 contribution significantly increases risk for PE in multigravidas who do not carry this allele, potentially mediated by a gradual maternal alloimmune response to repeated exposure to the paternal HLA-G variant.