The Sysmex XN-1000V provides a percentage and concentration of basophils from the WNR scattergram, as for human samples, but this method has been shown to be irrelevant in cats. This study aimed to characterize the feline basophil distribution on the WDF channel and to preliminarily evaluate the performance of a new basophil gate on the WDF channel. Cases of feline basophilia were retrospectively retrieved and the scattergram from the WDF and WNR channels were evaluated for any consistent pattern. A new gating setting for the WDF channel was created to include the suspected basophil region. This new gating was applied retrospectively to identified basophilia cases and prospectively to randomly selected feline cases. Manual, WNR, and new WDF methods for basophil identification were compared. Nine cases of feline basophilia were identified. A characteristic WDF scattergram was identified in seven of the nine cases. A new gate was created on the WDF channel and applied to these and 34 additional cases. The comparison study showed that the new method of basophil quantification using the WDF scattergram correlated better with the manual method than the Sysmex XN-1000V method using the WNR scattergram. Basophil concentration in feline peripheral blood can be determined using a new gate on the WDF channel of the Sysmex XN-1000V, which provides better performance than the WNR channel and is comparable to the manual method.