Fundulus heteroclitus, or mummichogs, are small (6-10 cm) teleost fish that are common in salt marshes, bays and estuaries along the East Coast of North America. The distribution of this species is such that populations in the extreme north experience a mean annual temperature of 6°C while those in the south are exposed to a mean annual temperature of 20°C, a thermal gradient of roughly 1°C per degree of latitude. F. heteroclitus are extremely polymorphic and several metabolic enzyme-encoding loci (Ldh-B, Mdh-A, Gpi-B, Idh-A, Est-S) have been found to vary greatly between northern and southern populations forming distinct clines. It has been suggested that gene frequency variance in isozymes may be a compensatory response to temperature. The non-catalytic ion binding protein, parvalbumin (PV), is thought to increase the rate of relaxation in fast twitch muscle fibers through its high affinity for calcium (Ca++). The purpose of this study was to determine if PV from F. heteroclitus would also exhibit clinal variance between northern and southern populations in response to the steep temperature gradient. The amino acid sequence for the major isoform of PV was determined from four populations of F. heteroclitus collected from the East Coast of the United States. These populations represented the observed gene frequency based clinal variation between northern and southern populations of F. heteroclitus. The dissociation constants (KD) of this PV isoform binding to Ca++ and Mg++ were determined for a temperature regime of 0-25°C. Additionally, the amino acid sequence from a sister species, Fundulus grandis, the Gulf killifish, and an outgroup, Fundulus similis, the Longnose killifish, were obtained and compared to F. heteroclitus to examine divergence within the genus. Due to the northerly invasion of F. heteroclitus it was expected that PV from northern populations would maintain function at lower temperatures than southern populations. The major isoform expressed in all populations of F. heteroclitus was identical by amino acid sequence analysis, indicating the absence of clinal variance in the expressed gene. Ion binding function of for this PV isoform is relatively insensitive to temperature. KD values for PV binding to Ca++ and Mg++ at 0, 5, 15, and 25°C were 7.88 ± 1.073, 7.77 ± 0.5, 7.64 ± 0.633, and 8.25 ± 0.55 and 0.24 ± 0.014, 0.32 ± 0.086, 0.36 ± 0.027, and 0.16 ± 0.019 mM, respectively.