The soil pathogenic fungus Phytophthora cactorum causes the most dangerous diseases occurring in strawberry plantations—strawberry crown rot and leather rot. Modern biotechnology methods, e.g., in vitro culture selection and molecular diagnostics can be utilized in the selection of cultivars that are less susceptible or resistant to Phytophthora diseases. In this study, in vitro selection of four strawberry microclones: ‘Elsanta’, ‘Feltar’, ‘Teresa’ and ‘Plena SVdT’ against Phytophthora cactorum (Lebert and Cohn) J. Schröt was carried out. Molecular analysis with inter simple sequence repeat (ISSR) markers was also used to evaluate genetic similarity of the selected resistant plants. None of the analyzed microclones showed complete resistance to the selection factor, but there were plants in all tested microclones that survived the pressure of the pathogen. Results showed that susceptibility to this pathogenic fungus was significantly differentiated and depended on the microclone. The ‘Feltar’ microclone had the significantly lowest susceptibility to Phytophthora disease, followed by the microclones ‘Elsanta’ and ‘Teresa’ with significantly higher susceptibility. The ‘Plena SVdT’ microclone showed the highest susceptibility to Phytophthora disease. This differentiation was linked to the genetic similarity observed at deoxyribonucleic acid (DNA) level between the resistant plants selected from microclones. Cluster analysis revealed that microclones with similar susceptibility to phytophthorosis, i.e., ‘Elsanta’, ‘Feltar’ and ‘Teresa’, appeared to be genetically similar. The microclone ‘Plena SVdT’ revealed a different course of phytophthorosis from the aforementioned microclones, being the least genetically similar to them.