The genus Phytophthora contains more than 120 species which have been classified in 10 clades based on morphological characteristics and molecular phylogenetic data. Many of these species pose a serious threat to horticulture (e.g. P. ramorum), agriculture (e.g. P. infestans) and natural ecosystems such as riparian areas, forests and heathlands (e.g. P. x alni). Of only a limited number of species the nuclear DNA content has been analysed by flow cytometry or estimated based on genome sequencing. Flow cytometry is the standard method for genome size analysis as genome sequencing in general underestimates genome sizes due to problematic sequencing at the centromeres and flaws in repetitive regions. Large genomes might indicate abundance of repetitive elements but can also be the result of genome duplication. Phytophthora hybrids might have a large DNA content due to auto- or allopolyploidisation (i.e. respectively polyploidisation after hybridization within or between species). The objective of this study was to determine the nuclear DNA content of Phytophthora isolates using an optimized protocol. Culture conditions were crucial to obtain high quality results. The genome size of over 90 Phytophthora isolates of about 30 species that belong to 7 different phylogenetic clades was determined using laser flow cytometry. Nuclei were extracted from mycelium and analysed simultaneously with an internal standard, either Arabidopsis thaliana (Col-0; genome size = 0.32 pg or 313 Mbp/2C) or Raphanus sativus cv. Saxa (genome size = 0.56 pg or 1086 Mbp/2C). Genome sizes were calculated relatively compared to the internal standard. After overnight staining with propidium iodide, nuclei were loaded on a flow cytometer and measured in triplicate on three different days using fresh Phytophthora cultures. The median genome size of the Phytophthora species measured was 179 Mbp and the average coefficient of variation calculated upon the three measurements per isolate was 4.0%. The smallest genome (112 Mbp) belonged to a P. kernoviae isolate while one isolate of P. x alni had the largest genome (839 Mbp). We included P. x serendipita and P. x alni hybrids in our study. The genome size of P. x serendipita was similar to that of both the parental species (P. cactorum and P. hedraiandra), indicating these might be sexual hybrids. The genome size of P. x alni was large, in line with its allopolyploid nature. Genome size analysis should be helpful in the identification and characterization of polyploid Phytophthora hybrids, which might form an increased pathogenic risk to crops and natural habitats.