ABSTRACT The objective of this study was to optimise the chromosome preparation technique by examining the impact of various factors on the quality of chromosome slices obtained from Newhall navel orange leaves. The wall removing hypotonic flame drying method was employed, and factors including leaf parts, developmental period, pretreatment time, enzyme ratio, and enzyme treatment duration were examined. Our findings revealed that the use of the apices of leaves from stage II, pretreated with 0.002 mol/L 8-hydroxyquinoline for 3 hours at room temperature, followed by enzymatic digestion in a mixture of 5% cellulase and 0.3% pectinase for 3 hours at 37 °C, provided the optimal conditions for chromosome preparation. This optimised protocol allowed for clear visualisation of morphological characteristics, establishing a reliable and efficient method for chromosome preparation in Newhall navel oranges.