Abstract Biological colouration presents a canvas for the study of ecological and evolutionary processes. Enduring interest in colour-based phenotypes has driven, and been driven by, improved techniques for quantifying colour patterns in ever-more relevant ways, yet the need for flexible, open frameworks for data processing and analysis persists. Here we introduce pavo 2 , the latest iteration of the R package pavo . This release represents the extensive refinement and expansion of existing methods, as well as a suite of new tools for the cohesive analysis of the spectral and (now) spatial structure of colour patterns and perception. At its core, the package retains a broad focus on (a) the organisation and processing of spectral and spatial data, and tools for the alternating (b) visualisation, and (c) analysis of data. Significantly, pavo 2 introduces image-analysis capabilities, providing a cohesive workflow for the comprehensive analysis of colour patterns. We demonstrate the utility of pavo with a brief example centred on mimicry in Heliconius butterflies. Drawing on visual modelling, adjacency, and boundary strength analyses, we show that the combined spectral (colour and luminance) and spatial (pattern element distribution and boundary salience) features of putative models and mimics are closely aligned. pavo 2 offers a flexible and reproducible environment for the analysis of colour, with renewed potential to assist researchers in answering fundamental questions in sensory ecology and evolution.