A simple protocol to extract RNA from a single wingless aphid, Aphis gossypii and Aphis spiraecola, using liquid nitrogen and Tri-Reagent is described. A nested reverse-transcription polymerase chain reaction with degenerate primers enabled Citrus tristeza virus (CTV) detection in these aphid species collected from an infected tangor ‘Ortanique’ (Citrus × sinensis (L.) Osbeck × Citrus reticulata Blanco) in the field. The procedure enabled a CTV detection rate of 75% in the A. gossypii and A. spiraecola evaluated.