Background: Acute diarrhea is one of the most serious problems in global public health that causes considerable morbidity and mortality worldwide. Human caliciviruses (HuCV) including norovirus (NoV, genogroup GI and GII) and sapovirus (SaV), is a leading cause of acute sporadic diarrhea in individuals across all age groups. However, few studies had been conducted clarifying the characteristics of HuCV in diarrhea cases across all age groups in China. Our study was aimed at assessing the HuCV-related diarrhea burden and NoV genotypes distribution in southwest China. Methods: The study was conducted in four hospitals in Kunming city, Yunnan province, from June 2014 to July 2015. Stool specimens were collected from 1,121 diarrhea cases and 319 healthy controls in outpatient departments. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect NoV (GI, GII) and SaV. Sequencing was applied to confirm the three viral infections and phylogenetic analysis was performed to determine their genotypes. A structured questionnaire was used to record the demographic information and clinical symptoms of subjects. Results: HuCV was detected at an 11.0 % infection rate in 1,121 diarrhea cases and at 3.4 % rate in 319 non-diarrhea subjects (p < 0.0001, OR = 3.5, 95 % CI 1.8–6.5). The prevalence of the NoV genogroup GII and genotype GII.4 in diarrhea cases was significantly higher than that found in healthy controls (p < 0.0001, p = 0.018, respectively). NoV GII (n = 118, 10.5 %) was the most common HuCV subtype in diarrhea cases, followed by SaV (n = 3, 0.3 %) and NoV GI (n = 2, 0.2 %). Of 118 NoV GII strains isolated from diarrhea patients. GII.4 (n = 55, 46.6 %) was the predominant strain, followed by GII.3 (n = 28, 23.7 %), GII.12 (n = 25, 21.2 %), GII.17 (n = 8, 6.8 %), and GII.5 (n = 2, 1.7 %). Of the 55 GII.4 strains, the GII.4 Sydney 2012 variant had absolutely predominant prevalence (n = 52, 94.5 %), followed by the NoV GII.4-2006b variant (n = 3, 5.5 %). The GII.4 Orleans 2009 variant was not found in diarrhea cases of the study. Conclusions: NoV GII was the major genogroup and GII.4 was the most predominant strain detected in diarrhea patients. The GII.17 is an emergent variant in sporadic diarrhea and might become the predominant strain in diarrhea cases in the near future. Rapid, accurate detection kits need to be developed to help us find and treat NoV-associated diarrhea in clinical settings in a timely manner.