CRISPR-Cas9 technology has been shown to be a very powerful tool to generate targeted mutations in a wide range of species. To evaluate its applicability for standard gene functional analysis in Petunia, we performed a case study aimed at obtaining loss-of-function alleles for six different Petunia genes. Using single guide RNAs (sgRNA), we obtained between 1 to 3 putative null alleles for 5 out of the six genes. Here we describe in detail the obtained results and the protocol used in this study, and discuss some of the technical problems encountered. Although the efficiency of the protocol should be further increased, these results demonstrate that CRISPR-Cas9 technology represents a very useful addition to the available molecular toolkit in petunia.