Some Neoparamoeba strains are pathogens of fish and invertebrates and were implicated in lobster mortality in Long Island Sound in recent years. To better understand the dynamics of these pathogens and their potential linkages to the mortality of marine animals, the capability to specifically detect and accurately quantify these parasites in the environment and in affected organisms is essential. Molecular markers such as mitochondrial cytochrome b (cob) can be powerful tools. In this study, we isolated cob from N. aestuarina and developed species-specific primers. Sequence analysis showed that this gene was A/T rich. Variable regions were identified and used to develop a species-specific primer set for real-time quantitative PCR (RTQ-PCR). This primer set was demonstrated to be specific (with no cross-reaction to N. pemaquidensis and other protists) and sensitive (with a detection limit of 1 cell/30 mL). Further, the quantitative capability of the RTQ-PCR system was verified by a strong correlation between the threshold cycle number and the logarithmic-transformed cob copy number used in the amplification. Using the established RTQ-PCR assay, we estimated that each N. aestuarina cell contained approximately 707 ± 63 copies of cob. This assay was applied to Lugol-fixed water samples collected from Long Island Sound during August 2002 to September 2003, which showed no detectable N. aestuarina. The cob primer set developed will be useful for future environmental detection of N. aestuarina and provides a basis from which cob gene probes for other Neoparamoeba and Paramoeba species can be developed.