The risk of malaria infection displays spatial and temporal variability that is likely due to interaction between the physical environment and the human population. In this study, we performed a spatial analysis at three different time points, corresponding to three cross-sectional surveys conducted as part of an insecticide-treated bed nets efficacy study, to reveal patterns of malaria incidence distribution in an area of Northern Guatemala characterized by low malaria endemicity. A thorough understanding of the spatial and temporal patterns of malaria distribution is essential for targeted malaria control programs. Two methods, the local Moran's I and the Getis-Ord G*(d), were used for the analysis, providing two different statistical approaches and allowing for a comparison of results. A distance band of 3.5km was considered to be the most appropriate distance for the analysis of data based on epidemiological and entomological factors. Incidence rates were higher at the first cross-sectional survey conducted prior to the intervention compared to the following two surveys. Clusters or hot spots of malaria incidence exhibited high spatial and temporal variations. Findings from the two statistics were similar, though the G*(d) detected cold spots using a higher distance band (5.5km). The high spatial and temporal variability in the distribution of clusters of high malaria incidence seems to be consistent with an area of unstable malaria transmission. In such a context, a strong surveillance system and the use of spatial analysis may be crucial for targeted malaria control activities.