Four polymorphic enzymes (PEP, PGI, PGM and IDH) were separated from adult individuals of five aphid parasitoid species of the genus Aphidius Nees (A. ervi Haliday, A. microlophii Pennacchio & Tremblay, A. eadyi Starý, Gonzalez & Hall, A. picipes Nees and A. urticae Haliday) using horizontal cellulose acetate plate electrophoresis. These markers were used to investigate the genetic relationships, including reproductive isolation and host adaptation/specificity, in laboratory and field populations. Samples were collected from the pea aphid, Acyrthosiphon pisum (Harris) and/or the nettle aphid, Microlophium carnosum (Buckton) in the UK and Bulgaria between 1991 and 1994. Whilst all loci discriminated between some species, PGM discriminated all five species, one species (A. eadyi) bearing two unique alleles (PGMa and PGMc). Aphidius microlophii (from nettle aphid) and A. ervi (from pea aphid), which are difficult to separate morphologically, possessed unique PGM alleles – PGMb and PGMe, respectively. Both parasitoids occur sympatrically, and whilst hybrids heterozygous for PGM were produced in the laboratory (PGMb,e), such genotypes were not observed in the field populations sampled. Hence, the species appear to be reproductively isolated. Most parasitoid populations studied showed mean heterozygote deficiencies per locus (homozygote excess) compared with Hardy-Weinberg expectations. In particular, A. eadyi bearing PGMa alleles were always homozygous whilst additionally, many were homozygous for another allele, PGIb. This is evidence for the existence of one or more morphologically-indistinguishable ‘cryptic’ species occurring sympatrically within European field populations. A dendrogram of relatedness was produced following calculation of Nei's genetic identity coefficient, I from the parasitoid population allele frequency data. All species showed very high similarity between populations at the intraspecific level (>0.9), but fewer interspecific similarities (0.23–0.63). These values compare well with previously published values for Aphidius populations and for other insects.