Rudbeckia L. are valuable nursery crops that offer broad adaptability and exceptional ornamental merit. However, there is little information on interspecific and interploid crossability and ploidy levels of specific cultivars. The objectives of this study were to determine the ploidy levels and relative DNA contents (genome sizes) of selected species and cultivars, to evaluate self-compatibility and crossability among species and ploidy levels, and to explore reproductive pathways in triploid R. hirta L. with the goal of facilitating future breeding endeavors and development of new hybrids. Reciprocal interspecific crosses were performed between R. hirta cultivars and R. fulgida Ait., R. missouriensis Engelm. ex C.L. Boynton & Beadle, and R. subtomentosa Pursh. as well as reciprocal interploid crosses among four R. hirta cultivars. A combination of relative DNA content analysis and chromosome counts was used to test for hybridity and to determine ploidy levels for selected species, cultivars, and interploid R. hirta F1 hybrids. Of the specific clones tested, R. subtomentosa and R. missouriensis were diploid, R. fuligida varieties were tetraploid, and R. hirta include both diploid and tetraploid cultivars. Mean 1Cx DNA content varied over 320% among species. The interploid R. hirta crosses produced triploids as well as pentaploids and hexaploids. Seedlings from open-pollinated triploid R. hirta appeared, based on diverse phenotypes and DNA contents, to be aneuploids resulting from sexual fertilization, not apomixis. Of the 844 seedlings from interspecific F1 crosses, only one individual, R. subtomentosa ×R. hirta, had a DNA content intermediate between its parents and was confirmed as the only interspecific hybrid. Although most taxa had low self-fertility, seedlings (with genomic sizes similar to their maternal parent) resulted after interspecific crosspollination, indicating that pseudogamy is one reproductive pathway in Rudbeckia species.