As with many crops, leaf chlorophyll fluorescence (LCF) emission is a promising tool for measuring responses of sugarcane to biotic and abiotic stresses particularly because LCF can be easily measured using portable fluorometers. The purpose of this presentation is to evaluate the effects of leaf measurement surface, number of measurements, and dark-period duration on sugarcane LCF measurements with a portable fluorometer. Four replications of eight sugarcane genotypes were planted in pots filled with organic (muck) soil, sand soil, or sand soil with organic matter added. Small leaf sections were cut from young leaves and LCF was measured after dark durations in the laboratory of 20 minutes, 3 hours, or approximately 18 hours. Storage was in paper bags for leaf sections exposed to the 20-minute dark duration. The 3- and 18-hour measurements were taken after storage in petri dishes with or without moistened filter papers. Measurements of LCF were taken on the abaxial and adaxial sides and on each side of the midrib on three leaf sections from each treatment. Measuring LCF on each side of the midrib did not improve precision. Broad sense heritability estimates were unacceptably low for the 20-minute dark duration and were similar for the 3- and 18-hour dark durations. One measurement of LCF should be taken per excised leaf section and leaves among treatments should be measured on the same side of the leaf (abaxial or adaxial) after at least a 3-hour dark period.