The 29-year-old star, who was replacing comedian Chelsea Handler as a guest host on her show Chelsea Lately, didn't hold back in ridiculing her nearest and dearest on live television.
"My mum basically tags around with Kim, follows her around, dresses like Kim," laughed Khloé, as a photo flashed up of the mother-daughter duo striding along the street, both wearing all-white outfits.
"It's like the Kim and Kris syndrome — they've started dressing alike," she added to fits of laughter from the audience. "I'm her favourite today, but normally it's Kim every day, all day, every day."
Kris Jenner and Kim Kardashian matching on a previous occasion
Opening the show, Khloé alluded to her "hot" sister Kim, who is engaged to rapper Kanye West.
"In case you've been to the checkout line at a grocery store, my family and I tend to appear in a lot of magazines," said Khloé. "I know this might be shocking, but all the stories they print are 100 percent false. Well, 99 percent are false."
"One of the tabloids recently said that I am hotter than Kim now," she joked. "Let's hashtag that 'winning.'"
Khloé, who is separated from her husband Lamar Odom, looked relaxed and confident as she took the reins for Chelsea's show. The Keeping Up With The Kardashians star was completely at ease joking about her body image and fighting off rumours that she was pregnant.
"I can confirm that I'm not pregnant," said Khloé. "If I were, my boobies would actually exist. They're so flat right now!"
The television personality showed no signs of nerves before going live on air, as she posed backstage in a series of sultry photos.
"Cause even forever ain't forever," she captioned a photo montage, in which Khloé is pictured in a revealing champagne-coloured dress that showed off her amazing curves.
"Are you my mother @heathermcdonald ?? Tune into@chelsealately_e tonight at 11pm," wrote Khloé alongside another photo. The sassy star posed in between her mother and comedian Heather McDonald, who looks just like Kris wearing a cropped wig.
For the rest of the show Khloé filled Chelsea's shoes and kept her audience entertained, joking about rumours surrounding her parentage and being in a "romantic relationship" with the regular host.