The three-dimensional architecture of DNA within the nucleus is a key determinant of interactions between genes, regulatory elements, and transcriptional machinery. As a result, differences in loop structure are associated with differences in gene expression and cell state. Here, we introduce diffloop, an R/Bioconductor package for identifying differential DNA looping between samples. The package additionally provides a suite of functions for the quality control, statistical testing, annotation and visualization of DNA loops. We demonstrate this functionality by detecting differences in DNA loops between ENCODE ChIA-PET datasets and relate looping to differences in epigenetic state and gene expression.