Environmental officials in Andalusia reported on Monday that a female Iberian lynx had been found dead on a roadway heading towards the town of Vilches.
The lynx had been tracked by a radio transmitter and monitored by local environmental researchers.
Another female lynx was found dead in Marmolejo in southern Spain five days earlier, also run over by a car.
This lynx was not fully grown and had not been tracked by researchers.
Many on social media mourned the loss of the endangered felines.
"Intolerable: Yesterday another female lynx was run over in Jaén, this one with a transmitter," wrote environmental journalist José Antonio Montero. "It is distressing to witness this blood."
"Another linx run over, this time in Marmolejo Jaén. Indefensible!" wrote animal rights group EQUO.
"Bad news... A lynx was killed by being run over in Marmolejo," tweeted the World Wide Fund for Nature, Spain. "We have to continue improving the highways."
The Iberian lynx is one of the world's most endangered cats and Spain has made great strides to boost its population in the wild since it was nearly wiped out at the end of last century.
Andalusia announced plans in December to monitor the wild cats using drones.
Illegal hunting, loss of habitat and diseases that have killed their natural prey of rabbits caused the number of lynx to plummet by the end of the 1990s to just 94 in two small pockets of Andalusia.
But progressive captive breeding programmes that released more into the wild and efforts to protect the cats' habitat grew the lynx population to more than 300 by the end of 2014.
Scientists confirmed at the end of last year that the lynx had made a comeback to Madrid after 40 years.
In recognition of the huge efforts made, the Iberian lynx was downgraded from a 'critically endangered' species to 'endangered' on the International Union for the Conservation of Nature (IUCN) Red List when it was updated in July.