Genome sizes were measured and determined for the karyotypes of nine species of aphid parasitoids in the genus Aphelinus Dalman,1820. Large differences in genome size and karyotype were found between Aphelinus species, which is surprising given the similarity in their morphology and life history. Genome sizes estimated from flow cytometry were larger for species in the Aphelinus mali (Haldeman, 1851) complex than those for the species in the Aphelinus daucicola Kurdjumov, 1913 and Aphelinus varipes (Förster,1841) complexes. Haploid karyotypes of the Aphelinus daucicola and Aphelinus mali complexes comprised five metacentric chromosomes of similar size, whereas those of the Aphelinus varipes complex had four chromosomes, including a larger and a smaller metacentric chromosome and two small acrocentric chromosomes or a large metacentric and three smaller acrocentric chromosomes. Total lengths of female haploid chromosome sets correlated with genome sizes estimated from flow cytometry. Phylogenetic analysis of karyotypic variation revealed a chromosomal fusion together with pericentric inversions in the common ancestor of the Aphelinus varipes complex and further pericentric inversions in the clade comprising Aphelinus kurdjumovi Mercet, 1930 and Aphelinus hordei Kurdjumov, 1913. Fluorescence in situ hybridization with a 28S ribosomal DNA probe revealed a single site on chromosomes of the haploid karyotype of Aphelinus coreae Hopper & Woolley, 2012. The differences in genome size and total chromosome length between species complexes matched the phylogenetic divergence between them.