Taking to Twitter, the 53-year-old branded Meghan "delusional", saying she "missed out a very important detail" in her speech.
"Dad paid her college tuition!" Samantha wrote, "She forgot the most important part of her speech and there are receipts to prove it.
"I love my sister but this is ridiculous. You can't lie like this, Meg! Delusionally absurd."
Meghan's speech stated, "I am also fully aware of the challenges of being able to afford this level of schooling for many people around the world – myself included.
"It was through scholarships, financial aid programmes and work-study where my earnings from a job on campus went directly towards my tuition - that I was able to attend university. And, without question, it was worth every effort."
However, Samantha remains adamant Thomas Markle paid for Meghan to attend university.
Thomas has previously revealed that he funded Meghan's education.
Speaking to Mail on Sunday, Markle said he sent his daughter to exclusive private schools from kindergarten onwards and he paid for her $30,000-a-year tuition at Northwestern University, Illinois, with $750,000 winnings from a lottery.
Royal fans were quick to pick up on the duchess' supposed dig at her father.
One wrote, "Did Meghan just clap back to her Liar sister and disgraceful father in that speech without even mention their names?"
Another added: "She did just that. With such a talent and grace, too."
Meghan has reportedly cut off her father and half-sister from her new royal life, as they continue to give countless media interviews.