Aiming to develop a new stratification protocol for yerba mate seeds, we evaluated how different combinations of temperature, substrate, and period influence the embryonic development of the species, compared to fresh seeds and conventionally stratified seeds. We measured the seeds’ moisture content and performed a tetrazolium test to evaluate embryo viability and embryonic development. Seeds’ initial moisture content was 10.1% and reached 40.6% after stratification. We observed more embryos in advanced development stages after stratification in all treatments, indicating that the protocols used were adequate for the species and have a great potential to be explored for yerba mate seedling.