Around 180,000 passengers will be affected by the walkout, Lufthansa said Monday, after last-minute talks with pilots' union Vereinigung Cockpit failed to reach a wages deal.
A spokesman for the group told AFP it would seek an injunction from a Munich labour court to block the strike, although a similar request was rejected by a Frankfurt court last week and the airline lost a second time at appeal.
Pilots have been battling the German behemoth since April 2014 over pay.
But pilots stuck to their demand of a pay rise of an average of 3.66 per cent per year, retroactive for the past five years.
The latest round of industrial action, which started on Wednesday, is the 14th since April 2014.
While Tuesday's walkout will only affect short-haul flights from Germany, Wednesday will see some long-haul services also affected.
Lufthansa said the flight plan for Tuesday and Wednesday would bring the number of passengers affected over the week of strikes to more than 525,000, with 4,461 cancelled flights.
The airline reached a pay and conditions deal with cabin crew in July after they staged the firm's longest-ever walkout in November 2015.
That seven-day stoppage saw 4,700 flights cancelled and more than half a million passengers grounded.
The deal with cabin crew also included a no-strike agreement until 2021 in exchange for job guarantees.
Lufthansa in October said it expected annual earnings before interest and tax in 2016 to reach "approximately the same level" as 2015's 1.8-billion-euro (USD 1.9 billion) figure.
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