Walther Flemming was born in Sachsenberg, Mecklenburg, now in Germany. He was a military physician during the Franco-Prussian War. Flemming held positions at the University of Prague (1873-76), and at the University of Kiel (1876-1901).

Flemming was one of the first to devote his time to cytology, the study of chromosomes. Cell division had been described as early as 1842 by Carl Nageli, who thought it was an anomalous event. Flemming was the first to detail the chromosomal movements in the process of mitosis. In 1879, Flemming used aniline dyes, a by-product of coal tar, to stain cells of salamander embryos. He was able to visualize the threadlike material as the cells divide. He described the whole process in his book Zell-substanz, Kern und Zelltheilung (Cell-Substance, Nucleus, and Cell-Division), which was published in 1882. Much of what we know today about mitosis originated with Flemming's observations. He saw that chromosomes were "doubled" when they appeared in prophase, and "solved" the problem of chromosomal partitioning between mother and daughter cells. This was significant for later work in meiosis and the chromosomal theory of inheritance.