The Pepino mosaic virus (PepMV) is one of the most important pathogens infecting tomato crops worldwide. Four major genotypes have been identified so far: Peruvian (LP), European (EU), Chilean 1 (CH1) and Chilean 2 (CH2). In Poland isolates representing the EU and CH2 genotypes have been found. The isolates belonging to the CH2 genotype induce a wide range of symptoms from very mild mosaic or even a lack of symptoms, through severe yellowing, up to necrosis of leaves. Therefore, the characterization of different genetic variants of PepMV is important to clarify processes of disease emergence caused by this virus. The necrotic and yellowing pathotypes representing the Chilean 2 genotype have been extensively studied. Development of infectious clones facilitates manipulation of the viral genome by site-directed mutagenesis. The studies performed made it possible to identify the viral genetic determinants responsible for the induction of different symptoms on infected plants. The introduction of single nucleotide substitutions in particular positions of triple gene block 3 (TGB3) resulted in changes of symptoms on tomato plants. Moreover, the mutations affect significantly viral accumulation, which has been measured by real-time RT-PCR. Single nucleotide substitutions introduced to the genome of genetically different European isolates also had an effect on biological properties and accumulation of the virus. Identified mutations in the viral genome are responsible for the development of symptoms and virus accumulation, and may play an important role during infection.