The aim of the presented study was to determine the effect of chemical mutagens on the morphological characteristics in M3 generation of petunia. The morphological changes referred principally to: discoloration of pigments in the flowers, as well as chlorophyll changes on the leaves and a return of crown-shaped petals. The results showed that EMS and DES at a dose of 0.5 mM were the most effective for inducing mutations. ISSR-PCR techniques were used to assess changes at the DNA level. The highest diversity among the analyzed genotypes of M3 generation of petunia were obtained after the amplification (GA)8A, (AC)8G, (GA)8T and (AC)8C (3) primers.