Parthenogenesis, meaning “creation by virgin,” is a key component of apomixis (asexual reproduction through seeds). It refers to spontaneous embryogenesis from an unfertilized egg cell resulting in offspring that is genetically identical to the mother plant. The diplosporous apomictic grass Tripsacum dactyloides (Tripsacum) represents a valuable natural model system for studying gene expression patterns associated with parthenogenesis. All investigated polyploid Tripsacum accessions reproduce via facultative apomixis with parthenogenetic frequencies of >90%. Here, we report a strategy and method regarding how parthenogenesis genes can be identified from a non-model species whose genome is not yet available. We describe the manual dissection of parthenogenetic and sexual egg cells from polyploid and sexual Tripsacum accessions, respectively, the generation of RNA-seq data from few cells and their mapping to a closely related species (here maize) whose genome is known. We further describe a comparative approach to identify candidate parthenogenesis genes and how their parthenogenetic potential and the reproduction mode can be elucidated in transgenic approaches.