Arising from the putative chorionic-type intermediate trophoblast, epithelioid trophoblastic tumor is a recent addition to the spectrum of gestational trophoblastic diseases. Frequently, the tumor involves the uterine cervix and is misdiagnosed as invasive squamous-cell carcinoma. The pathogenesis of the tumor is poorly understood, and its molecular analysis is essentially lacking. This study was designed to explore chromosomal alterations in epithelioid trophoblastic tumor and to use DNA genotyping to demonstrate its trophoblastic origin, therefore separating the tumor from its mimics of the maternal origin. Five cases of epithelioid trophoblastic tumors were included in this study and paired DNA samples from the tumor and normal tissue were extracted from paraffin-embedded archival materials. The status of chromosomal alterations was analyzed by comparative genomic hybridization using conventional metaphase chromosome preparations. The parental genetic contribution was determined by DNA genotyping analysis using AmpFISTR Identifiler Amplification system (Applied Biosystems Inc.). Comparative genomic hybridization analysis was successful in three cases analyzed, all of which showed a balanced chromosomal profile without detectable gain or loss of the genome. DNA genotyping was informative in four epithelioid trophoblastic tumor involving anatomic locations including the cervix (two cases), endomyometrium (one case) and lung (metastatic, one case). All four cases were found to have unique paternal alleles, confirming the trophoblastic nature of the tumors. In summary, chromosomal alterations detectable by conventional comparative genomic hybridization are not features of epithelioid trophoblastic tumors. In difficult cases, the presence of the paternal alleles demonstrated by DNA genotyping is a powerful diagnostic application in separating an epithelioid trophoblastic tumor from its maternal mimics, particularly the far more common squamous-cell carcinoma of the uterine cervix.