Cluster 1 streptokinases (SK1) from Streptococcus pyogenes (GAS) show substantially higher human plasminogen (hPg) activation activities and tighter hPg binding affinities than cluster 2b streptokinases (SK2b) in solution. The extent to which the different domains of SK are responsible for these differences is unknown. We exchanged each of the three known SK domains (α, β, and γ) between SK1 and SK2b and assessed the function of the resulting variants. Our results show that primary structural differences in the β-domains dictate these functional differences. This first report on the primary structure–functional relationship between naturally occurring SK1 and SK2b sheds new light on the mechanism of hPg activation by SK, a critical virulence determinant in this species of human pathogenic bacteria.