The rocket was the first of its kind to be fired at the city of 122,000 since informal cease-fires were declared separately by Israel and Hamas two weeks ago at the end of Israel's bruising three-week-long offensive in Gaza.
No one was injured in Tuesday's attack, police said.
Israel launched its "Operation Cast Lead" offensive on Dec. 27 to halt near-daily rocket fire from Gaza at Israel targets.
Sporadic rocket and mortar fire have continued, however, reports CBS News correspondent Robert Berger, testing the fragile Gaza cease-fire.
Israel has responded with air strikes targeting Hamas militants and weapons smuggling tunnels and has threatened disproportionate retaliation, but is holding off because national elections are just a week away, Berger adds.
More than a dozen rockets and mortar shells slammed into Israel on Sunday. The following day Israel fired a missile at a car in the town of Rafah, killing a Palestinian militant, and bombed the nearby Gaza-Egypt border, seeking to destroy tunnels that Hamas uses to smuggle in weapons and supplies.
Foreign Minister Tzipi Livni pledged to keep hitting Hamas as long as it attacks Israel and ruled out negotiations with the militant Islamic rulers of Gaza.
"Terror must be fought with force and lots of force. Therefore we will strike Hamas," she said at a security conference Monday. "If by ending the operation we have yet to achieve deterrence, we will continue until they get the message."
Continued violence could work against Livni's government in the Feb. 10 general election and bolster hard-line opposition leader Benjamin Netanyahu, who is seen as the front-runner.
Ashkelon was hit by nearly 100 rockets during the Gaza fighting. Following Tuesday's rocket attack, a local parents union called for classes to be called off. But city hall announced that school would be open as usual.