Three synthetic triploid seedlings were derived from a cross between maternal parent Populus × euramericana (Dode) Guinier and Populus nigra var. italica (Moench.) Koehne via embryo rescue by treating female floral buds with colchicine at certain developmental stages before and after pollination, and identified by flow cytometric analyses. A total of 21 microsatellite primers were used to infer the ploidy of gametes forming the hybrids and the underlying meiotic mechanism. The results suggest that 2n eggs originated from the maternal poplar, contributing the extra haploid genome in the triploids. In addition, first division restitution (FDR) 2n eggs were produced in the maternal tree according to the marker genotypes of these triploids. Cytological analyses of megasporogenesis revealed that meiosis of the maternal parent was asynchronous. This cytological evidence suggests that 2n eggs result from FDR after treatment with colchicine, as a portion of megaspore mother cells (MMCs) were within the first division at the post-pollination stage. These results aid in understanding the mechanism of 2n egg formation in poplar and make the utilization of 2n eggs in polyploid poplar breeding more effectively.