The common estuarine fish species Fundulus heteroclitus or Mummichog has long been used in ecotoxicological studies; however, their periodic spawning pattern is perceived to limit their use in reproductive bioassays and the availability of sensitive life stages. This study evaluated the lunar periodicity of spawning, egg production, and changes in gonadosomatic index (I_G) in the laboratory using wild and F₁-generation Mummichogs that originated from three different geographic areas: the northern extreme part of their geographic range (New Brunswick, Canada; 45°N), the middle of the northern subspecies range (Massachusetts, USA; 41°N), and within the southern subspecies range (Virginia, USA; 37°N). Unlike some previous studies of wild and laboratory-held fish, Mummichogs from all locations were continuous spawners over a prolonged period of time (8 weeks) under laboratory conditions. However, there was greater variance in egg production in smaller fish (≤70 mm total length) compared to larger fish. Our results demonstrate the potential to use Mummichogs consistently in testing protocols due to the absence of lunar spawning in the laboratory, and further suggest that larger fish should be used to optimize the power to interpret results when conducting reproductive bioassays.