Giardia duodenalis is one of the most frequent intestinal parasitic infections in children worldwide. To date, eight main assemblages of G. duodenalis have been described, but only A and B genetic groups are known to infect humans. A cross-sectional study was conducted to determine the prevalence and risk factors of Giardia duodenalis infection in 417 preschool children from the Fomento municipality in the central region of Cuba between January and June 2013. The overall prevalence of Giardia infection was 10.79%. Assemblage identification was carried out by the amplification of a fragment of the triose phosphate isomerase (tpi) gene. DNA from 36 of 45 (80%) samples was successfully amplified by PCR-tpi. Assemblage B and mixed assemblages A+B represented 52.78 and 36.11% respectively, of genotyped samples. Assemblage A accounts for only 11.11%. Children who were cared for at home were associated with diarrhea caused by assemblage B. No associations were found between other clinical variables with infecting assemblage of Giardia. Univariate analysis identified the use of unsafe water resources (OR 2.9; CI 1.2-6.8) and-even more interestingly-keeping dogs indoor (OR 2.5; CI 1.2-5.4) were significant risk factors associated with Giardia infection among children. Multivariate analysis using introduction test logistic regression ratified the association of these two risk factors: kept dogs indoor (OR 2.8, CI 1.1-5.3), and untreated water (OR 1.4, CI 1.4-4.9) with Giardia infection. This information may be useful for an effective prevention and control programme of giardiasis in this population.