Viral cross-protection in plants is a phenomenon, where a mild virus isolate can protect plants against damage caused by a severe challenge isolate of the same virus. It has been used on a large scale in cases where no resistant plants are available. We examined differences in cross-protection between pathotypes of Pepino mosaic virus representing Chilean 2 genotype. The potential of a mild PepMV-P22 isolate to protect tomato against more aggressive challenge isolates causing yellowing and necrotic symptoms was established. The challenge isolates were PepMV-P5-IY (yellowing), PepMV-P19 (necrotic) and PepMV-P22 K67E (artificial necrotic mutant of PepMV-P22 which differ from PepMV-P22 only by a point mutation). Efficient cross-protection was obtained using mild PepMV-P22 against PepMV-P5-IY. After a challenge inoculation with PepMV-P19 or PepMV-P22 K67E symptoms severity were significantly reduced in comparison to non-protected plants; however, necrotic symptoms appeared two months after coinfection. The real-time PCR analysis revealed that the level of accumulation of the necrotic isolate in tomato plants was even 5–7 times higher than that of PepMV-P22.