Parentage testing in the domestic dog is finding increasing application for dog breed registries as well as in research. The aim of parentage verification is the correct assignment of both parents to the offspring. For accurate parentage verification informative microsatellite markers have to be identified. More powerful models to study artificial insemination in bitches will be possible if the paternity and gender of early dog conceptuses can be determined. The amelogenin gene locus has been used in bovine day six to day seven embryos for early gender determination but no research has been done on early conceptuses of the domestic dog. The aim of the current study was to establish an accurate method for parentage and gender determination from domestic dog conceptuses during early pregnancy in a multi-sire insemination trial. Semen from 10 male dogs was used in each of 12 females for artificial insemination. Blood and uterine tissue for DNA extraction was collected from males and females, and embryonic material was collected after ovariohysterectomy between 16 and 30 days after the onset of cytological dioestrus. Twenty-three microsatellite markers were used for parentage verification, and the amelogenin gene locus for gender determination. Mean observed heterozygosity, mean expected heterozygosity (HExp), and mean PIC were high (0.6753, 0.6785, and 0.628, respectively). There were 66 conceptuses. In two, neither parentage nor gender could be established because their tissue samples were contaminated. Parentage could be assigned by CERVUS 3.0.3 in 42 out of 64 of the cases (66%) without difficulty. Another 33% of the cases (21 out of 64) could be resolved using the number of exclusions, LOD scores or manual verification of genotyping errors. In one conceptus, paternity could not be established because its sire may have been either of two siblings. The gender of the female and male dogs was successfully confirmed using the amelogenin gene locus. The gender of the conceptuses was determined using the amelogenin gene locus (50% male, 50% female) but not confirmed by another method of gender determination such as karyotyping. As shown in the current study, the panel of 24 microsatellite markers used provides high information content suitable for parentage verification in multi-sire litters, as well as gender determination of early conceptuses.