Barbara was born in Kassel, Hessen on 8 April 1536, one of the ten children of Philip I, Landgrave of Hesse and his legitimate wife Christine of Saxony.

She had four sisters and five brothers including George I, Landgrave of Hesse-Darmstadt.

Her paternal grandparents were William II of Hesse and Anna of Mecklenburg-Schwerin, and her maternal grandparents were George, Duke of Saxony and Barbara Jagiellon, daughter of King Casimir IV Jagiellon of Poland and Elisabeth of Austria.

They made their residence at the Chateau de Montbeliard in the principality of Mömpelgard, a staunch Lutheran enclave in France.

Among her numerous descendants are the current Spanish and British Royal Families.