Three-spined stickleback populations are also found in freshwater lakes and streams. These populations were probably formed when anadromous fish started spending their entire life cycle in freshwater, and thus evolved to live there all year round. Freshwater populations are extremely morphologically diverse, to the extent that many observers (and some taxonomists) would describe a new subspecies of three-spined stickleback in almost every lake in the Northern Hemisphere. One consistent difference between freshwater populations and their anadromous ancestors is the amount of body armour, as the majority of freshwater fish only have between 0 and 12 lateral armour plates, and shorter dorsal and pelvic spines. However, there are also large morphological differences between lakes. One major axis of variation is between populations found in deep, steep sided lakes and those in small, shallow lakes. The fish in the deep lakes typically feed in the surface waters on plankton, and often have large eyes, with short, slim bodies and an upturned jaw. Some researchers refer to this as the limnetic form. Fish from shallow lakes feed mainly on the lake bed, and are often long and heavy bodied with a relatively horizontal jaw and a small eye. These populations are referred to as the benthic form.
Many populations take 2 years to mature and experience only one breeding season before dying and some can take up to 3 years to reach maturity. However, some freshwater populations and populations at extreme latitudes can reach maturity in only 1 year. In spring, males defend territories where they build nests on the bottom of the pond or other body of water; the sequence of territorial, courtship and mating behaviours was described in detail by Niko Tinbergen in a landmark early study in ethology. Territorial males develop a red chin and belly colouration, and Tinbergen showed that the red colour acted as a simple sign stimulus, releasing aggression in other males and the first steps in the courtship sequence from gravid females. Red colouration is produced from carotenoids found in the diet of the fish. As carotenoids cannot be synthesised de novo, the degree of colouration gives an indication of male quality, with higher quality males showing more intense colouration. However, it is noteworthy that the response to red is not universal across the entire species complex, with black throated populations often found in peat-stained waters. Males also develop blue irises on maturation. Only the males care for the eggs once they are fertilised. Parental care is intense, involving nest maintenance and fanning of the eggs to ensure a fresh water supply, even at night. Males build the nests from vegetation, sand, pebbles and other debris, adhering the material together with spiggin, a proteinaceous glue-like substance secreted from the kidneys. Sticklebacks have four colour photoreceptor cells in their retina, making them potentially tetrachromatic. They are capable of perceiving ultraviolet wavelengths of light invisible to the human eye and use such wavelengths in their normal behavioural repertoire.
The three-spined stickleback is a known intermediate host for the hermaphroditic parasite Schistocephalus solidus, a tapeworm of fish and fish-eating birds.