Groundwater ecosystems are very diverse in respect to biodiversity and physical environment. These open systems are strongly connected to the open water as well as adjacent terrestrial and atmospheric ecosystems. Groundwater taxa represent a large array of biota including viruses, prokaryotes (bacteria and archaea), microeukaryotes (fungi and protists) and metazoans. Their activities sustain groundwater ecosystem functions (e.g., cycling of material and flow of energy), and the resulting ecosystem services (e.g. purification of water) significantly contribute to human well-being. Research on the ecology and evolution of groundwater organisms dates back to the early 1900s. Our current understanding ranges from biodiversity to biogeography, from genes to mechanisms involved in adaptation and development of specific biological traits, from the inter- and intraspecific interactions to carbon and energy flow through food webs, and from physical–chemical and structural drivers to the role of individual organisms in groundwater ecosystem functioning and services. This research has been providing the basis for integrating ecosystem functions and resulting services into the governance and management of groundwater, thereby generating joint benefits to society and biodiversity. The second edition of “Groundwater Ecology and Evolution” is composed of 24 chapters grouped into six sections that portray the diversity of groundwater research conducted by ecologists and evolutionary biologists. This includes, but is not restricted to, the hydrogeological and hydrochemical attributes of groundwater habitats, the controls and patterns of groundwater biodiversity, the role of organisms in groundwater systems, the evolutionary processes and forces for acquisition of subterranean biological traits, and the way these traits are differently expressed among organisms. Finally, it discusses the challenges and opportunities for conservation of groundwater biodiversity and management of groundwater ecosystems.