Compared with single hemispheresegment, hemispheresegment group has better forward and reverse unequal fluid resistance properties. In order to analyze the impact of flow resistance variation on pumping performance, the changes of row number, column number and intervals for hemispheresegment group in pump chamber were studied. Forward and reverse flow resistance coefficient formulas of hemispheresegment group were derived and verified by the flow resistance experiment and flow rate experiment of the pump. In the range of limited row and column intervals, the experimental and theoretical flow resistance difference and flow rate increased along with the increase of row and column numbers. At the driving voltage of 120V and the driving frequency of 6Hz, the valveless piezoelectric pumps with hemispheresegment group in rows and columns 3×4, 4×3, 4×4 respectively obtained the pump flow rates of 45.5mL/min, 46.2mL/min, 47.75mL/min The maximum deviation of theoretical relative to experimental flow rate was 23.23%. The results showed that the flow resistance law of hemispheresegment group could be used to analyze and predict the pump flow rate; the pump output flow rate can be increased by increasing the row number or column number meanwhile properly limiting the row interval and column interval. The better output performance can be obtained by increasing row number than column number.