Ranking amongst the most economically damaging pests of soybean [Glycine max (L.) Merr.], the soybean cyst nematode (SCN, Heterodera glycines Ichinohe) threatens soybean production on a global scale. The primary strategy for managing SCN infestation is by planting high-yielding resistant cultivars. The objective of this study was to critically evaluate cultivars from the Cianzio soybean breeding project for yield and defensive traits developed from 2000 to 2020 combining high seed yield and SCN resistance. Forty-nine genotypes including checks were tested during 2017 and 2018 seasons on two field sites each year, one on SCN-infested and another on non-infested soil. The genotypes were additionally evaluated in controlled greenhouse conditions. Agronomic and SCN-related disease resistance traits were scored for all genotypes, and molecular profiling was done to determine the source of SCN resistance. Results indicate the program successfully introgressed SCN resistance genes, using three novel sources in addition to Peking and PI 88788, all protecting yield. All resistant genotypes, verified by qPCR rhg1 copy number estimates, proved to effectively control nematode reproduction and subsequent nematode population numbers at the SCN-infested location. This study has confirmed the use of the simple sequence repeat Satt309 molecular marker as an effective method for selecting for SCN resistance and determining rhg1 copy number repeats.