Objective: To describe prenatal diagnosis of hemoglobin (Hb) Bart’s hydrops fetalis caused by a previously undescribed condition in Thailand of the interaction of αo-thalassemia with the Southeast Asian (–SEA) and the THAI (–THAI) deletions in a Thai family. Methods: Molecular and hematological features associated with αo-thalassemia carriers found in a pregnant Thai woman and her husband were investigated. Prenatal diagnosis was performed at her third pregnancy on amniotic fluid using a multiplex PCR for simultaneous detection of the (–SEA) and (–THAI) αo-thalassemia determinants. Results: Initial DNA analysis for the common form of αo-thalassemia (–SEA) identified that the pregnant woman was a carrier whereas her husband was negative for this mutation. They had a healthy non-thalassemic daughter from the first pregnancy but the second pregnancy ended unexpectedly with Hb Bart’s hydrops fetalis. Analysis of the archived DNA specimen of the husband demonstrated that he was in fact a carrier of the (–THAI) αo-thalassemia determinant. A successful application of a multiplex PCR for simultaneous detection of these two forms of αo-thalassemia in prenatal diagnosis at the third pregnancy was demonstrated and identified that the fetus was a carrier of the (–SEA) αo-thalassemia. Conclusion: Although relatively rare, the (–THAI) αo-thalassemia should be included in a routine screening to prevent a Hb Bart’s hydrops fetalis syndrome. A simple DNA assay based on a multiplex PCR approach would therefore provide an effective means for αo-thalassemia screening in the region.