The short petiole trait is valuable for the development of plant ideotype with high yield. Soybean breeding line, SS98206SP, showed extremely short petioles in greenhouse and field. In this study, the short petiole of two mutant lines, SS98206SP and D76-1609, were investigated to determine the genetic segregations. These two mutants were crossed with each other and with two normal petiole genotypes. Genetic analysis indicated that the short petioles in D76-1609 and SS98206SP were controlled by a single recessive gene, respectively. The short petiole gene in SS98206SP was non-allelic with lps, conferring short petiole in D76-1609. Two recessive genes showed complementary relationship having short petioles with recessive homozygote (LPS1-lps?lps?, lps1lps1LPS?-, lps1lps1lps?lps?). Our data indicated that the short petioles in SS98206SP were controlled by a single recessive gene designated as lps3.