The early life-history requirements of Polyodon spathula (paddlefish) are not well understood, in part due to the difficulty of sampling early life stages. Passive sampling with benthic, mat-style devices effectively collected paddlefish eggs in the Tombigbee watershed (Mobile River basin) during spring 2005, facilitating identification and characterization of egg-incubation microhabitats. Eggs were collected over gravel, sand-impacted gravel, and bedrock substrates at corrected depths ranging from 1.2 to 7.7 m. Sampling occurred continuously (489 sampler-days) in the lotic bendway of the Tennessee-Tombigbee Waterway from late February through April, when water temperatures ranged from 11.5 to 20.8 °C. Of 106 paddlefish eggs collected from this unique macrohabitat, 95% were taken on either April 6 or April 16. Nine paddlefish eggs were collected in a tributary (Noxubee River) on April 13 after four sampler-days of effort. Water temperatures associated with peak spawning activity ranged from 16.9 to 19.4 °C, slightly higher than temperatures recorded for Mississippi River basin populations. A substantial (> 2.74-m) rise in water level triggered spawning activity, similar to that observed in other systems. Benthic mats proved useful for delineating paddlefish egg-incubation habitat in areas not subject to shifting substrate, and could be used in the future to address hypotheses regarding micro- and macrohabitat suitability.