三人行必有我妹是一部动画片，为了继承父亲帝野熊五郎（小山力也 配音）的遗嘱，高中生帝野将悟（樱井孝宏 配音）进入了贵族学校深流院学园学习如何成为一个合格社长，不仅如此，在接下来的两年中，将悟还必须在学园中找到可以托付一生的结婚伴侣。这可难倒了个性普通的将悟，不过，世上无难事，将悟愉快的高中生活就要开始了。 在生日的当天，将悟接到了一个自称是“妹妹”的女孩打来的电话，然而这位妹妹究竟是谁，将悟毫无头绪。在学园中，将悟结识了许多可爱的女孩子，有学级委员鹤真心乃枝（石原夏织 配音），沉默害羞的神凪雅（佐仓绫音 配音），高智商少女嵯峨良芽衣（日高里菜 配音）等人，而将悟感觉到，那个神秘的妹妹，就藏在这些花朵般柔软的女孩子们之中。
Is insanity hereditary? Shougo Mikadono's beginning to think so, because the terms of his late father's will seem crazy and following them may drive Shougo bonkers as well. Oh, it sounds simple at first: before Shougo can claim his VERY large inheritance, he just has to start attending a certain new school and find a nice girl to marry. It's a little unromantic, but perfectly do-able, right? After all, all the girls seem quite friendly, so all Shougo has to do is find one he has something in common with. EXCEPT, and here's the kicker, it turns out that Shougo has WAY too much in common with one of them, because she's actually his long lost sister. And he has no idea which one she is. Will Shougo meet and court his Miss Right without committing something very morally wrong? Can he find his future bride without slipping into the wrong set of genes? And if his little sister does reveal herself, just how much will be revealed and under what circumstances?
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