Dissolved calcium (Ca2+) concentrations in freshwater ecosystems are of growing concern as increasing levels have been implicated in altering the environmental conditions and biodiversity. Elevated Ca2+ levels and sporadic re-emergence and disappearance of invasive zebra mussels in Kentucky Lake in recent years served as a motivation for this study. The objective of this study was to determine Ca2+ spatial and temporal patterns in Kentucky Lake, selected tributary streams, and the Ohio River during and following a zebra mussel invasion. Over 1000 water samples were collected and analysed for dissolved calcium during 2018-2022. Approved analytical methods were followed for sampling and measuring dissolved calcium levels. Results revealed significant spatial and temporal patterns. Kentucky Lake Ca2+ levels varied between 15-25 mg/L depending on the sampling location and month/year. Kentucky Lake channel sites exhibited comparably higher concentrations of Ca 2+ than did most embayment and/or stream sites, indicating that tributary streams did not serve as primary sources of calcium to the lake. Dissolved calcium levels at main lake sites exceeded the threshold for zebra mussel growth and reproduction in 2018 during the time when zebra mussels were present. Calcium in lake water samples collected from 2019 through 2022 was at or just below the threshold. Temporal trend data showed a gradual increase in Ca2+ in Kentucky Lake throughout the study period but remaining at or below the threshold level considered critical for the zebra mussels’ reproduction and development. Calcium levels in the Ohio River site at Paducah were similar to Kentucky Lake reflecting the predominance of Tennessee River water, while levels at the Brookport site were consistent with values known to support zebra mussel populations. The elevated calcium levels in Kentucky Lake waters during the late winter and early spring months may be due to natural sources (mineral weathering) as well as human activities in the Tennessee River basin. This study emphasizes the need for continued calcium monitoring in the watershed to determine the potential for future zebra mussel outbreaks and potential influences on the lake ecosystem and its functions.