Noroviruses (NoVs) are a leading cause of acute gastroenteritis outbreaks and sporadic cases of diarrhea in industrialized countries. To study the prevalence and genetic diversity of NoVs in Guatemala, stool specimens were collected from hospitalized and ambulatory patients presenting with diarrhea (≥3 loose or liquid stools in a 24-hr period) who were enrolled in a prospective surveillance system in the Departments of Santa Rosa (October 2007 to August 2010) and Quetzaltenango (August 2009 to August 2010), Guatemala. Specimens were tested for rotavirus, enteric bacteria, and parasites by routine methods and for genogroups I and II NoV by real-time reverse transcription-PCR. A total of 2,403 stool specimens were collected from hospitalized (n = 528) and ambulatory patients (n = 1,875). Overall, 341 (14%) samples tested positive for NoVs including 114 (22%) hospitalized and 227 (12%) ambulatory patients. NoVs disease peaked during the winter (November–January) months. Among the 341 NoVs-positive patients, 32 (9%) were also positive for rotavirus, 32 (9%) for bacteria, and 9 (3%) for protozoa. Nucleotide sequences were obtained from 84 samples collected from hospitalized children aged <5 years of age, which could be grouped into nine GII and three GI genotypes with GII.4 (74%) and GI.8 (10%) being the most common. This is the first study on the prevalence of NoVs among hospitalized and ambulatory patients with diarrhea in Guatemala. The findings highlight the need to implement laboratory diagnostics for NoVs to improve appropriate clinical management of diarrheal diseases and guide vaccine development.