Abstract: Flow cytometry method is capable of facilitating improvement in a long gestation crop like cashew through characterization and screening of parent plants, evaluation of population biodiversity, screening of hybrids and ploidy analysis of induced mutants. Unfortunately, report on the application of flow cytometry to cashew (Anacardium occidentale) in the literatures is rare thus limiting information on its genome. To develop a flow cytometric protocol amenable to the plant, five different buffer compositions, three sample sizes and five incubation periods of isolated nuclei were evaluated using six cashew accessions. Six plant species commonly used as internal reference standards were also screened to find most suitable reference for cashew. Data collected on the quality of nuclear suspension in terms of fluorescence intensity, background yield (%), nuclear yield (nuclei sec-1 mg-1) and coefficient of variation (%) of G0/G1 peak were summarized and statistically analysed using analysis of variance and relevant descriptive statistics procedure. The results showed high background noise from the flow cytometric analysis of the isolated nuclei, an indication that cashew plant contains high content of pehnols and cytostil compounds. Among the buffer systems examined, cashew performed better with Otto buffer. Preparation from leaf tissue of about 70 mg incubated for about 10 min on cold ice in the dark gave satisfactory results. Tomato, Solanum lycopersicum cv. Stupicke with 1.96 pg/2C genome was found most ideal internal reference for determination of nuclear DNA content in cashew. The protocol reported here would be useful for ploidy analysis and estimation of relative nuclear DNA content and not absolute genome size in cashew.