In July 2013, disease symptoms were observed on cassava crop, including longitudinal lip-like fissures on roots, and a thick cork-like appearance on root peel. The diseased cassava contained very low starch content. No symptoms were observed on leaves or stems. In Paraguay, the frogskin disease has been detected in the Central Department. Recently, typical symptoms were also observed in the San Pedro Department. The 16SrIII-L phytoplasma has been associated with the disease in Colombia. A total of 59 symptomatic roots and 10 asymptomatic samples were collected from the San Pedro Department and total DNA was extracted. Based on the results of nested polymerase chain reaction (PCR), real-time PCR assay and nucleotide sequence analysis, the causal agent was identified as a 16SrIII phytoplasma. This is thought to be the first report of a phytoplasma associated with cassava frogskin disease in Paraguay. Symptoms were observed in most of the samples, indicating the possible emergence of this disease in the country.