Experimental and field studies of different turtle species suggest that moisture influences embryonic development and sex ratios, wetter substrates tend to produce more males, and drier substrates produce more females. In this study, we used Trachemys scripta elegans to test the effect of moisture on embryonic development and sex ratios. T. s. elegans eggs were incubated under different temperature and moisture regimes. We monitored embryonic development until stage 22 (after sex determination) and, for the first time, we estimated sex ratios using a male-specific transcriptional molecular marker, Sox9. Among treatments, we found differences in developmental rates, egg mass, and sex ratio. Embryos developed slowly in cooler and wetter sand substrate while water uptake by the eggs was significantly greater on wetter substrates. Developmental differences were due to moisture interacting with temperature where increased water content of the sand resulted in temperatures that were 2–3 °C lower than air temperatures. The coolest and the wettest substrates produced 100% males compared to 42% males from the warmest and driest treatment. Further, we found that embryonic growth appears to be more sensitive to temperature at earlier stages of development and to moisture at later stages. This study shows how moisture may change the incubation conditions inside nests by changing the temperature experienced by eggs, which affects development, growth and sex ratios. The results of this study highlight the importance of including moisture conditions when predicting embryo growth and sex ratios and in developing proxies of embryonic development.