Rotavirus is the most common cause of acute gastroenteritis among infants and young children throughout the world, but rotavirus cases in developing countries account for nearly all of the ∼ 600,000 annual deaths. We studied the epidemiology of rotavirus in 22 rural communities in northern coastal Ecuador over a five-year period. From 250 rotavirus positive stool specimens, the percentage that could not be RT-PCR genotyped for VP4 and VP7 was 77% and 63%, respectively. The possibility of sample degradation was considered but discounted after an experimental examination of rotavirus stability and EM visualization of rotavirus-like particles in several untypeable samples. Finally, alternate primers were used to amplify Ecu534, a sample that was untypeable using most published VP4 and VP7 primers. Characterization of the VP7, VP4, and VP6 full gene segments revealed novel genotypes and nucleotide mismatches with most published primer sequences. When considered with other findings, our results suggest that primer mismatch may be a widespread cause of genotyping failure, and might be particularly problematic in countries with greater rotavirus diversity. The novel sequences described in this study have been given GenBank accession numbers EU805775 (VP7), EU805773 (VP4), EU805774 (VP6) and the RCWG has assigned them novel genotypes G20PI13, respectively.