Maximal oxygen uptake (VO₂max) has been traditionally used to explain physiologic differences among soccer teams of different ranking. However, other endurance markers may have greater discriminatory ability. The purpose of this study was to examine whether velocity at lactate threshold and running economy can be used to better discriminate endurance characteristics of soccer teams of different levels along with VO₂max during preseason testing. One hundred twenty-nine professional Greek soccer players participating in the top 3 divisions underwent an incremental treadmill test to exhaustion using expired gas analysis and simultaneous blood lactate measurements. Velocity at lactate threshold was determined using the Dmax method, and running economy was measured at 12 km·h⁻¹. Analyses of variation were used to compare for differences between divisions. Velocity at lactate threshold was the only variable that was statistically different between any 2 divisions. In every comparison, the higher division had the higher velocity at lactate threshold. The VO₂ was statistically different only between the top 2 divisions. Running economy was statistically different between divisions with similar VO₂, with better running economy for the higher division in each comparison. These results indicate that velocity at lactate threshold can be used to better discriminate endurance characteristics of soccer teams of different level along with VO₂max during preseason testing. Running economy may reveal differences between teams with similar VO₂max.