The concern comes as charges have been filed against a Simi Valley man suspected of shooting and killing P-38, the incident being disclosed just days after another mountain lion's death.
The National Park Service estimates there are just 10 to 15 adult cougars in the local hills, and two of them have recently been killed.
Ana Beatriz Cholo, with the Santa Monica Mountains National Recreation Area, says the big cat labeled P-61 - a four year old male - may have been the cougar spotted in a Tarzana backyard over the summer.
P-61 was struck by a car over the weekend as it tried to cross the 405 Freeway.
"Freeways and wildlife typically don't mix," Cholo said. "They act like fences, and it's not great for the mountain lion population here."
The Ventura County District Attorney's Office revealed another mountain lion, P-38, was illegally shot and killed in July on the campus of The American Jewish University in Simi Valley.
Alfredo Gonzalez, 59, has been charged with killing a protected mammal and vandalizing its collar.
Eyewitness News was unable to reach Gonzales for comment.
A source tells us he had been employed by American Jewish University, but has since been terminated.
The school says it's cooperating with investigators and is "committed to working closely with the California Department of Fish and Wildlife, other relevant authorities, and community partners to ensure that AJU is fully prepared for any foreseeable situations involving wildlife in the future."
"What's sad is that he was also a male mountain lion who was breeding, so there aren't that many of them in the Santa Monica Mountains," said Cholo.
With so few of the big cats, experts worry about their genetic diversity, and the future strength of the cougar community, something Cholo says is a cherished rarity.
"We are able to sustain a mountain lion population within the city boundaries. Mumbai and L.A. are the only two megacities that have that," she said.
Meantime, wildlife officials are excited about a planned bridge over the 101 Freeway. The wildlife crossing would provide cougars and other animals a way to safely move above traffic.
Construction is expected to start within the next two years.