With its estrogenic activity, ( S )-equol plays an important role in maintaining host health and preventing estrogen-related diseases. Exclusive production occurs through the transformation of soy isoflavones by intestinal bacteria, but the reasons for variations in ( S )-equol production among different individuals and species remain unclear. Here, fecal samples from humans, pigs, chickens, mice, and rats were used as research objects. The concentrations of ( S )-equol, along with the genetic homology and evolutionary relationships of ( S )-equol production-related genes [daidzein reductase (DZNR), daidzein racemase (DDRC), dihydrodaidzein reductase (DHDR), tetrahydrodaidzein reductase (THDR)], were analyzed. Additionally, in vitro functional verification of the newly identified DDRC gene was conducted. It was found that approximately 40% of human samples contained ( S )-equol, whereas 100% of samples from other species contained ( S )-equol. However, there were significant variations in ( S)- equol content among the different species: rats > pigs > chickens > mice > humans. The distributions of the four genes displayed species-specific patterns. High detection rates across various species were exhibited by DHDR , THDR , and DDRC . In contrast, substantial variations in detection rates among different species and individuals were observed with respect to DZNR . It appears that various types of DZNR may be associated with different concentrations of ( S)- equol, which potentially correspond to the regulatory role during ( S)- equol synthesis. This enhances our understanding of individual variations in ( S )-equol production and their connection with functional genes in vitro . Moreover, the newly identified DDRC exhibits higher potential for ( S)- equol synthesis compared to the known DDRC, providing valuable resources for advancing in vitro ( S)- equol production., Importance: ( S )-equol (( S )-EQ) plays a crucial role in maintaining human health, along with its known capacity to prevent and treat various diseases, including cardiovascular diseases, metabolic syndromes, osteoporosis, diabetes, brain-related diseases, high blood pressure, hyperlipidemia, obesity, and inflammation. However, factors affecting individual variations in ( S )-EQ production and the underlying regulatory mechanisms remain elusive. This study examines the association between functional genes and ( S )-EQ production, highlighting a potential correlation between the DZNR gene and ( S )-EQ content. Various types of DZNR may be linked to the regulation of ( S )-EQ synthesis. Furthermore, the identification of a new DDRC gene offers promising prospects for enhancing in vitro ( S )-EQ production., Competing Interests: The authors declare no conflict of interest.