Technical Abstract: Plants express three phylogenetic classes of hemoglobins (Hb) based on sequence analyses. Class 1 and 2 Hbs are full length globins with the classical 8 helix Mb-like fold, whereas Class 3 plant Hbs resemble the truncated globins found in bacteria. With the exception of the specialized leghemoglobins, the physiological functions of these plant hemoglobins remain unknown. We have reviewed and, in some cases, measured new the oxygen binding properties of a large number of Class 1 and 2 plant non-symbiotic Hbs and leghemoglobins. We found that sequence classification also predicts distinct extents of hexacoordination with the distal histidine and markedly different overall oxygen affinities and association and dissociation rate constants. These results suggest strong selective pressure for the evolution of distinct physiological functions. The leghemoglobins evolved from the class 2 globins and show no hexacoordination, very high rates of O2 binding (~250 µM-1s-1), little electrostatic stabilization of bound ligands, moderately high rates of O2 dissociation (~5-15 s-1), and high oxygen affinity (P50 ˜ 50 nM). These properties both facilitate O2 diffusion to respiring, N2 fixing bacteria and reduce O2 tension in the root nodules of legumes. The class 1 plant Hbs show weak hexacoordination (KHisE7 ˜ 2), moderate rates of O2 binding (~25 µM-1s-1), very strong electrostatic stabilization of bound O2, very small rates of O2 dissociation (~0.16 s-1), and remarkably high O2 affinities (Kd or P50 ˜ 2 nM), suggesting a function involving O2 and NO scavenging. The class 2 Hbs exhibit strong hexacoordination (KHisE7 ˜ 100), low rates of O2 binding (~1 µM-1s-1), moderate electrostatic stabilization of bound O2, moderately low O2 dissociation rate constants (~1 s-1), and moderate, Mb-like O2 affinities (Kd or P50 ˜ 340 nM), perhaps suggesting a sensing role for sustained low, micromolar levels of oxygen.