Tomato (Lycopersicon esculentum) is an important vegetable crop in Mexico. Recently, a phytoplasma associated with leaf yellowing and curling, severe stunting and little leaf in tomato plant was identified as Yucatan tomato phytoplasma (16SrIII group). DNAs extracted from tomato leaves with symptoms were examined for the presence of this phytoplasma by nested polymerase chain reaction (PCR). Positive results were obtained in 44% of samples, yielding an rDNA product of 1.25 kb. In vitro and in silico restriction fragment length polymorphism (RFLP) patterns obtained with endonucleases HpaII, MseI, RsaI and TaqI were characteristics of group 16SrIII, according to the classification scheme of phytoplasmas. The pattern with AluI and HaeIII discriminated between these phytoplasmas and the members of 16SrIII group. Molecular characterization of the causal agent of Yucatan tomato phytoplasma will facilitate the study of this disease’s epidemic aspects and its phytosanitary management. In addition, it will contribute to a greater knowledge of the genetic diversity of phytoplasmas present in Mexico.