Activated coagulation factor XIII (FXIIIa) cross-links the γ-chains of fibrin early in clot formation. Cross-linking of the α-chains occurs more slowly, leading to high molecular weight multimer formations that can also contain γ-chains. To study the contribution of FXIIIa-induced γ-chain cross-linking on fibrin structure and function, we created 2 recombinant fibrinogens (γQ398N/Q399N/K406R and γK406R) that modify the γ-chain cross-linking process. In γK406R, γ-dimer cross-links were absent, but FXIIIa produced a cross-linking pattern similar to that observed in tissue transglutaminase cross-linked fibrin(ogen) with mainly α-γ cross-links. In Q398N/Q399N/K406R, cross-links with any γ-chain involvement were completely absent, and only α-chain cross-linking occurred. Upon cross-linking, recombinant normal fibrin yielded a 3.5-fold increase in stiffness, compared with a 2.5-fold increase by α-chain cross-linking alone (γQ398N/Q399N/K406R). γK406R fibrin showed a 1.5-fold increase in stiffness after cross-linking. No major differences in clot morphology, polymerization, and lysis rates were observed, although fiber diameter was slightly lower in cross-linked normal fibrin relative to the variants. Our results show that γ-chain cross-linking contributes significantly to clot stiffness, in particular through γ-dimer formation; α-γ hybrid cross-links had the smallest impact on clot stiffness.