Much evolutionary theory assumes that parasite virulence (i.e. parasite-induced host mortality) is determined by within-host parasite reproduction and by the specific parasite genotypes causing infection. However, many other factors could influence the level of virulence experienced by hosts. We studied the protozoan parasite Ophryocystis elektroscirrha in its host, the monarch butterfly, Danaus plexippus. We exposed monarch larvae to wild-isolated parasites and assessed the effects of within-host replication and parasite genotype on host fitness measures, including pre-adult development time and adult weight and longevity. Per capita replication rates of parasites were high, and infection resulted in high parasite loads. Of all host fitness traits, adult longevity showed the clearest relationship with infection status, and decreased continuously with increasing parasite loads. Parasite genotypes differed in their virulence, and these differences were maintained across ecologically relevant variables, including inoculation dose, host sex and host age at infection. Thus, virulence appears to be a robust genetic parasite trait in this system. Although parasite loads and genotypes had strong effects on virulence, inoculation dose, host sex and age at infection were also important. These results have implications for virulence evolution and emphasize the need for a detailed understanding of specific host-parasite systems for addressing theory.