Citrus tristeza virus (CTV) was reported in the 1960’s to affect Meyer lemon trees in Chile, but no field symptoms were observed. This study performed a complete biological, serological and molecular characterization of one hundred CTV isolates obtained from different hosts and geographical origins. Decline symptoms (DI) were not found on trees grafted onto sour orange. In Pica Oasis in northern Chile, stem pitting (SP) was found to affect grapefruit and Mexican lime trees. Most isolates present in the central area were considered mild (MCA-13-negative), while in the northern area aggressive isolates were observed and detected. Some of these isolates were capable of causing SP on grapefruit under field and greenhouse conditions and on sweet orange under greenhouse conditions. Almost all of these isolates were MCA13-positive and had nucleotide sequences associated with the VT genotype