Globalization has provided opportunities for parasites/pathogens to cross geographic boundaries and expand to new hosts. Recent studies showed that Nosema ceranae, originally considered a microsporidian parasite of Eastern honey bees, Apis cerana, is a disease agent of nosemosis in European honey bees, Apis mellifera, along with the resident species, Nosema apis. Further studies indicated that disease caused by N. ceranae in European honey bees is far more prevalent than that caused by N. apis. In order to gain more insight into the epidemiology of Nosema parasitism in honey bees, we conducted studies to investigate infection of Nosema in its original host, Eastern honey bees, using conventional PCR and duplex real time quantitative PCR methods. Our results showed that A. cerana was infected not only with N. ceranae as previously reported [Fries, I., Feng, F., Silva, A.D., Slemenda, S.B., Pieniazek, N.J., 1996. Nosema ceranae n. sp. (Microspora, Nosematidae), morphological and molecular characterization of a microsporidian parasite of the Asian honey bee Apis cerana (Hymenoptera, Apidae). Eur. J. Protistol. 32, 356-365], but also with N. apis. Both microsporidia produced single and mixed infections. Overall and at each location alone, the prevalence of N. ceranae was higher than that of N. apis. In all cases of mixed infections, the number of N. ceranae gene copies (corresponding to the parasite load) significantly out numbered those of N. apis. Phylogenetic analysis based on a variable region of small subunit ribosomal RNA (SSUrRNA) showed four distinct clades of N. apis and five clades of N. ceranae and that geographical distance does not appear to influence the genetic diversity of Nosema populations. The results from this study demonstrated that duplex real-time qPCR assay developed in this study is a valuable tool for quantitative measurement of Nosema and can be used to monitor the progression of microsprodian infections of honey bees in a timely and cost efficient manner.