Citrus tatter leaf virus (CTLV) is a graft-transmissible capillovirus that causes the tatter leaf disease of citrus and bud union incompatibility between scion and trifoliate hybrid rootstocks which can result in serious economic losses. Although several CTLV detection protocols have been described, there is a need for a highly sensitive protocol that can be used in routine diagnostic tests for virus-free budwood certification programs. To address this need, a TaqMan® chemistry based real time reverse transcription-polymerase chain reaction (RT-qPCR) assay was developed to detect CTLV from citrus plants. The nucleotide sequence analysis based on full genomic sequences of eight CTLV isolates available from GenBank showed that 3′ region of the viral genome is more conserved among different CTLV isolates than the rest of the viral genome. A new set of primers and a TaqMan® probe were designed from a conserved 500 nt-long fragment at the 3′ end of the CTLV genome for the detection of CTLV by RT-qPCR. The newly designed primers for CTLV RT-qPCR assay had ca. 98.6% amplification efficiency and showed 100% CTLV detection rate when they were tested for previously known CTLV-positive trees infected with different CTLV isolates. The sequencing of 132 bp-long RT-qPCR amplicon followed by BLASTn search showed that it had 94% to 98% sequence identity to those of various CTLV isolates available in GenBank, confirming the specificity of the assay to reliably detect CTLV.