The development of a chemically-mutagenized population of Petunia hybrida could enable the identification of novel alleles for crop improvement. Conditions were determined for mutagenizing petunia with ethyl methanesulfonate (EMS), while minimizing deleterious effects on viability and fertility. A mutagenized population of the doubled haploid P. hybrida line ‘Mitchell Diploid’ was developed as a resource for TILLING. Three mutagenesis parameters were investigated in this study: the imbibition of seeds prior to EMS treatment, the EMS concentration, and EMS exposure time. Based on these results, the mutagenesis of 2000 petunia seeds for TILLING was conducted with a 12 hour imbibition followed by exposure to 0.1% EMS for 12 hours.