Pontecorvo was born in Pisa, Italy. Having graduated in agricultural sciences from the university in his native city of Pisa in 1928, he spent the following nine years in Florence, supervising the Tuscany cattle-breeding program. Political conditions caused him to leave Italy in 1938, his intention being to continue with similar work in Peru. However he first accepted an invitation to the Institute of Animal Genetics in Edinburgh, where he met the famous American geneticist Hermann Muller, another visitor at the institute. Under Muller's influence Pontecorvo became increasingly interested in pure genetics and together with Muller he devised an elegant method for investigating the genetic differences between species that produce sterile hybrids on crossing.

Pontecorvo remained in Edinburgh working on Drosophila species and gained his PhD in 1941. Two factors then prompted him to change from Drosophila genetics to fungal genetics, firstly the dire need for penicillin during World War II and secondly his interest in the structure and function of the gene, a topic more easily investigated in the fungi.

Pontecorvo's work on the fungus Aspergillus nidulans led to the discovery, with Joseph Roper, of the parasexual cycle in 1950. This cycle gives rise to genetic reassortment by means other than sexual reproduction and its discovery provided a method of genetically analyzing asexual fungi. Pontecorvo also put forward the idea of the gene as a unit of function, a theory substantiated by Seymour Benzer in 1955. Pontecorvo occupied the first chair of genetics at Glasgow University from 1955 until 1961, when he moved to the Imperial Cancer Research Fund. He retired in 1975.