Herbicides have become one of the most widespread weed-control tools in the world since their advent in the mid-20th century [1]. Nowadays, they are still being used in most conventional cropping systems in modern agriculture [2]. Unfortunately, the persistent use of herbicides is being threatened by the spread of herbicide resistance, a fast evolutionary process that took place a few years after their arrival into modern agriculture [2,3]. To safeguard their future use in agriculture, there is great interest in understanding the molecular mechanisms conferring resistance or predisposing weeds toward evolving herbicide resistance. Herbicide resistance is governed by target-site resistance (TSR) and non-target-site resistance (NTSR) mechanisms [4]. TSR-based resistance is caused by any gene alteration able to change the interaction with the encoded target protein/enzyme so that the herbicide is not able to sufficiently interfere with it to cause plant death. TSR mechanisms are usually better understood because there is a single well-known target gene, and therefore, they are monogenic [5]. On the other hand, NTSR mechanisms are those not involving the target protein and can decrease the herbicide arriving at the site of action (SoA) into an insufficient amount, so plants can survive; more rarely, any mechanism protecting plants from herbicide damage is also referred as NTSR [5]. NTSR mechanisms are rarely fully understood since they can be quantitative in nature and controlled by several genes (with each gene providing some level of resistance); in other words, NTSR-based resistance can be polygenic [4]. The increase in multiple herbicide resistance to different SoAs, mainly through enhanced metabolism, is of great concern [2]. Multiple herbicide resistance reflects an evolutionary process by which populations or plants can accumulate different resistance mechanisms (TSR and/or NTSR), conferring resistance to several SoAs [6]. Sadly, this process usually occurs because resistance to one SoA provokes switching to another SoA rather than reducing herbicide-selection pressure [7]. Among NTSR mechanisms, enhanced metabolism is the most threatening because, as a generalist mechanism, it can confer cross-resistance to dissimilar herbicide chemistries, even to those never used before [4]. Conversely, TSR is governed by specialist mechanisms, always specific to a single SoA [5]. This Special Issue was focused on the new well-characterized cases of herbicide resistance, both for TSR and/or NTSR (if a molecular basis is reported), as well as studies that identify new gene alterations conferring TSR or the genetic basis involved in NTSR. Both TSR and NTSR can also be divided into different mechanisms depending on their nature. Point mutations, altered expression, or codon deletion of the target-site gene are among the most reported types of TSR mechanisms [5]. NTSR mechanisms usually involve altered patterns of herbicide absorption, translocation, or metabolism. Herbicide-metabolism-based resistances are complex and often involve genes that are members of large gene families, including cytochromes P450 (P450) and Glutathione-S-transferases (GST) [4]. Therefore, this editorial focuses on the nature of the resistance mechanisms of the two major types, TSR and NTSR, described in each of the contributions to this Special Issue.