Methanotrophic bacteria utilize methane as their sole carbon and energy source. Studies of the model Type II methanotroph Methylosinus trichosporium OB3b have provided insight into multiple aspects of methanotrophy, including methane assimilation, copper accumulation, and metal-dependent gene expression. Development of genetic tools for chromosomal editing was crucial for advancing these studies. Recent interest in methanotroph metabolic engineering has led to new protocols for genetic manipulation of methanotrophs that are effective and simple to use. We have incorporated these newer molecular tools into existing protocols for Ms. trichosporium OB3b. The modifications include additional shuttle and replicative plasmids as well as improved gene delivery and genotyping. The methods described here render gene editing in Ms. trichosporium OB3b efficient and accessible.