A lawyer by training, Jimenez de Quesada was appointed chief justice of Santa Marta colony (on the north coast of Colombia), where he arrived in 1535 with the fleet of Pedro Fernandez de Lugo. In 1536 he was commissioned by de Lugo to command an expedition to explore southwards into the interior of Colombia. A land party under Quesada, with Hernan Perez de Quesada (his brother), Juan San Martin, Juan del Junco (as second in command) and Lazaro Fonte, struck south from Santa Marta, crossed the Rio Cesar, and arrived at Tamalameque on the Magdalena River. A support fleet of 6 (or 5) ships had also sailed from Santa Marta with 800 men to navigate the Magdalena. Only two of the vessels actually arrived at Tamalameque, and subsequently returned to Santa Marta with many of Quesada's men. Continuing up the Magdalena as far as La Tora (=Barrancabermeja), Quesada and his men ascended the Opon River into the cordillera, reaching the Opon hills, Chipata (near Velez) (March 1537) and the valley of the Suarez River. Passing Lake Fuquene and Lake Suesca, they reached Nemoncon and Zipaquira, but with only 166 men left alive (600 had perished en route). From there they entered the lands of the King Bocota (at the site of present Bogota). They renamed Bacata (the major city) as Santa Maria de Bogota, and attacked Tunja. Quesada remained in the region until the arrival of the expeditions of Sebastián de Benalcázar from Ecuador and Nikolaus Federmann from Venezuela in 1539. The three generals then agreed on the refoundation of the capital and promptly decided to submit their rival territorial claims to the arbitration of the crown. From Cartagena they sailed for Spain, where Quesada pressed his claim as governor. In this he was unsuccessful, the governorship of Popayan being awarded to Benalcazar, but Quesada did return (in 1549) with the honorific title of governor of El Dorado.
In 1568, at the age of 60, Quesada received a commission to conquer Los Llanos to the east of the Colombian cordillera. From Bogota in April 1569 with 400 Spaniards, 1500 natives, 1100 horses and 8 priests, he first descended to Mesetas on the upper Guejar River. There most of the livestock was destroyed by a grass fire. Quesada's expedition then moved to nearby San Juan de los Lllanos, where a course was set for east-southeast (by the guide Pedro Soleto), and maintained for the following two years. After a year or so some men returned with Juan Maldonado, reaching San Juan after six months with few survivors. Quesada eventually reached (San Fernando de) Atabapo at the confluence of the Guaviare and the Orinoco (in December 1571), any further movement requiring the construction of ships. He therefore dejectedly returned to Bogota, arriving in December 1572 with only 64 Spaniards, 4 natives, 18 horses and 2 priests. The expedition had been one of the most expensive disasters on record, and after a brief period of service in a frontier command Quesada retired to Huesca with what he could salvage of his fortune. He died in Mariquita in 1579, and his remains were taken to the cathedral at Bogota.