Species in the genus Asclepias (milkweeds; Apocynaceae) are known for their production of cardiac glycosides, defensive metabolites that are important ecologically as a chemical defense in several plant families and medicinally as a treatment for heart disorders. There is considerable variation in cardiac glycoside production among milkweed species. For instance, Asclepias curassavica (tropical milkweed) has a high cardiac glycoside concentration, particularly of nitrogen- and sulfur-containing (N,S) cardiac glycosides, but its close relative Asclepias incarnata (swamp milkweed) has very low concentrations of cardiac glycosides overall. The first step of cardiac glycoside biosynthesis, the conversion of campesterol to pregnenolone, is likely catalyzed by enzymes in the cytochrome P450 87A (CYP87A) family in the Apocynaceae. Therefore, transient A. curassavica gene expression experiments were performed in the model plant Nicotiana benthamiana to determine whether a CYP87A3 homolog and two highly expressed candidate genes from the CYP90B family could catalyze this reaction. In a separate study, experiments were performed to identify candidates for the incorporation of cysteine in the synthesis of the N,S-cardiac glycosides. Gene expression in A. curassavica and Asclepias syriaca (common milkweed), which produce these compounds, was compared with that of close relatives of each, A. incarnata and Asclepias tuberosa (butterfly weed), respectively, which contain far less or none. Genes that are more strongly induced in both A. curassavica and A. syriaca may be involved in the biosynthetic pathway for N,S-cardiac glycosides, and eight genes fitting these criteria with predicted function in cardiac glycoside, sulfur, and/or cysteine metabolism were identified. These candidates, along with the transcriptome assemblies performed in the process of their identification, will facilitate further investigation into the ecology and evolution of Asclepias species.