Our previous study showed that smoke derived from alfalfa (Medicago sativa) caused different germination responses compared with that from prairie hay (Festuca hallii) and wheat straw (Triticum aestivum), but the mechanism remained unclear. In this study, we used Salad Bowl lettuce (Lactuca sativa) as a quick bioassay to trace the active compounds in each of these three smoke solutions. Column chromatography and high performance liquid chromatography (HPLC) were used to separate and identify active fractions. Seeds of four species from Fescue Prairie were primed for 24 h at room temperature in darkness using serial dilutions of separated active fractions, as well as karrikinolide (KAR1). After priming, seeds were dried at room temperature in darkness for 7 days and subsequently incubated at 10/0°C or 25/15°C in 12 h light–12 h dark or 24 h darkness for 49 days. KAR1 was in the smoke made from prairie hay, and wheat straw, but was absent in alfalfa smoke. Priming in KAR1 solutions increased germination of three native species. Priming in highly concentrated KAR1 reduced radicle length of Cirsium arvense, the only non-native species. Even though KAR1 has the potential to enhance regeneration of native species in the Fescue Prairie, KAR1 is not universally present in smoke derived from different plant materials. Unknown compound(s) in smoke derived from legumes remain to be identified.