Tuesday's flight brings the total for all initial F-35 flights from the base to 47, a spokeswoman said.
The Marines plan more conventional flights of the F-35B planes in coming months, gradually expanding to short takeoffs and vertical landings and more complex aerial training, the Pentagon said. "It's another milestone in what we are calling our execution year," said Air Force Colonel Andrew Toth, commander of the 33rd Fighter Wing at the base.
The base also provides certification classes for future F-35 pilots and extensive training for maintenance personnel. The successful first F-35B flight comes a week after rising costs and delays forced Britain to reverse course and opt to buy the B version of the F-35, which does not require a catapult and arrester wires to be fitted to UK aircraft carriers.
F-35 pilot training was initially slated to begin last fall, but was delayed by the Pentagon after its chief tester raised concerns about the maturity of the new plane and its software. Formal training of four to six pilots at a time will begin once officials receive approval from the Air Education and Training Command, a step that will come only after more local area flights and an evaluation of the plane's operational use. The command says its timetable will be driven more by events than any specific dates, but officials have said they hope to begin pilot training this summer.
Officials were now starting to validate flight instructions and a training syllabus.
The military needs trained pilots and maintainers in order to start using the new warplanes operationally.
The Marines are likely to be the first of the US military services to declare them ready for "initial operational capability" because they do not plan to wait for more sophisticated software upgrades required by the other services. Meanwhile, 3,650 machinists and other union workers are in the fifth week of a strike at the Fort Worth, Texas, plant where Lockheed builds the F-35, and two military bases where it is tested.
Lockheed on Saturday said it had reached new contracts with two smaller unions at the Texas plant.
Lockheed spokesman Joe Stout said no negotiations were slated with the union at this point. Lockheed is developing the multirole stealth F-35 for the US military and eight international partners at a projected cost of around $396 billion.
Britain's BAE Systems is a key contractor on the project.
Other subcontractors include Northrop Grumman Corp and United Technologies Corp